TW202528611A - Composite polymeric materials, and products and methods of preparing the same - Google Patents

Abstract

The disclosure provides leather articles having composite layer coatings including optional mechanically engineered topographical features.

Description

Composite polymeric materials, products thereof and preparation methods

The present disclosure relates to composite polymeric materials, which in part include cellulose derivative coating compositions, optionally including silk fibroin or fragments thereof, and various additional agents, for coating various substrates.

Silk is a natural polymer produced by various insects and spiders. It consists of a filamentous core protein, filoprotein, and a gelatinous coating, sericin, made up of non-filamentous proteins. Silk fiber is lightweight, breathable, and hypoallergenic.

Embodiments of the present disclosure provide a complex comprising a first polymeric macromolecular species or polymer and a second polymeric macromolecular species or polymer. In some embodiments, a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer are physically and/or chemically entangled. In some embodiments, a portion of the first polymeric macromolecular species or polymer is physically and/or chemically cross-linked. In some embodiments, a portion of the second polymeric macromolecular species or polymer is physically and/or chemically cross-linked. In some embodiments, a portion of the first polymeric macromolecular species or polymer is chemically and/or physically integrated into a portion of the second polymeric macromolecular species or polymer. In some embodiments, a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer are inseparable. In some embodiments, a portion of the first polymeric macromolecular species or polymer and/or a portion of the second polymeric macromolecular species or polymer are cross-linked. In some embodiments, a portion of the first polymeric macromolecular species or polymer and/or a portion of the second polymeric macromolecular species or polymer are partially organized and/or crystallized. In some embodiments, a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer cannot be stratified. In some embodiments, a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer are self-assembled. In some embodiments, a portion of the first polymeric macromolecular species or polymer in the complex has a second structure that is different from the first structure of the first polymeric macromolecular species or polymer. In some embodiments, a portion of the second polymeric macromolecular species or polymer in the complex has a second structure that is different from the first structure of the second polymeric macromolecular species or polymer. In some embodiments, a portion of the first polymeric macromolecular species or polymer in the complex has a second structure that is different from the first structure of the first polymeric macromolecular species or polymer, and a portion of the second polymeric macromolecular species or polymer in the complex has a second structure that is different from the first structure of the second polymeric macromolecular species or polymer. In some embodiments, the first polymeric macromolecular species or polymer comprises a protein component. In some embodiments, the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein, and/or whey. In some embodiments, the first polymeric macromolecular species or polymer comprises a biodegradable polymer. In some embodiments, the first polymeric macromolecular species or polymer comprises one or more of the polyurethane components. In some embodiments, the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component, a poly(lactic- co -glycolic acid) (PLGA) component, or both. In some embodiments, the second polymeric macromolecular species or polymer comprises a cellulose and/or cellulose derivative component. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. In some embodiments, the cellulose derivative is ethyl cellulose. In some embodiments, the ethoxy content of the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%. In some embodiments, the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3. In some embodiments, the second structure of the cellulose derivative comprises less than 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises between about 5% and less than about 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises a degree of crystallinity between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 99%, or between about 90% and about 100%. In some embodiments, the second structure of the cellulose derivative comprises less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81 ... about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 58%, less than about 57%, less than about 56%, less than about 58%, less than about 57%, less than about 56%, less than about 59%, less than about 58%, less than about 57%, less than about 56 ... %, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than The crystallinity of the composite is less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%. In some embodiments, the w/w ratio between the first polymeric macromolecular species or polymer and the second polymeric macromolecular species polymer in the composite is between about 1:100 and about 100:1. In some embodiments, the w/w ratio between the first polymeric macromolecular species or polymer and the second polymeric macromolecular species or polymer in the complex is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:1 :21, about 78:22, about 77:23, about 76:24, about 75:25, about 74:26, about 73:27, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 6 6:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, About 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72 , about 27:73, about 26:74, about 25:75, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:85, about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99. In some embodiments, the w/w ratio between the first polymeric macromolecular species or polymer and the second polymeric macromolecular species polymer in the complex is about 10:1, about 10:2, about 10:3, about 10:4, about 10:5, about 10:6, about 10:7, about 10:8, about 10:9, or about 10:10. In some embodiments, the first polymeric macromolecular species or polymer is isotropically distributed across a cross-section of the complex. In some embodiments, the first polymeric macromolecular species or polymer is anisotropically distributed across a cross-section of the complex. In some embodiments, the concentration of the first polymeric macromolecular species or polymer closer to the first surface of the complex is higher than the concentration of the first polymeric macromolecular species or polymer closer to the second surface of the complex. In some embodiments, the first polymeric macromolecular species or polymer is substantially undetectable at the second surface of the complex. In some embodiments, the second polymeric macromolecular species or polymer is isotropically distributed across a cross-section of the composite. In some embodiments, the second polymeric macromolecular species or polymer is anisotropically distributed across a cross-section of the composite. In some embodiments, the concentration of the second polymeric macromolecular species or polymer closer to the second surface of the composite is higher than the concentration of the second polymeric macromolecular species or polymer closer to the first surface of the composite. In some embodiments, the second polymeric macromolecular species or polymer is substantially undetectable at the first surface of the composite substrate-coating interface. In some embodiments, the first surface of the composite is adhesive. In some embodiments, the second surface of the composite is adhesive. In some embodiments, the first surface of the composite is adhesive and the second surface of the composite is adhesive. In some embodiments, the first surface of the composite is adhesive and the second surface of the composite is non-adhesive. In some embodiments, the composite has increased water resistance compared to one of the following: i) a non-composite material comprising a first polymeric macromolecular species or polymer but not a second polymeric macromolecular species or polymer, ii) a non-composite material comprising a second polymeric macromolecular species or polymer but not the first polymeric macromolecular species or polymer, or iii) a non-composite material comprising a first polymeric macromolecular species or polymer and a second polymeric macromolecular species or polymer, wherein the polymeric macromolecular species or polymers are not physically and/or chemically molecularly entangled. In some embodiments, the composite has increased water vapor permeability compared to one of the following: i) a non-composite material comprising a first polymeric macromolecular species or polymer but not a second polymeric macromolecular species or polymer, ii) a non-composite material comprising a second polymeric macromolecular species or polymer but not the first polymeric macromolecular species or polymer, or iii) a non-composite material comprising a first polymeric macromolecular species or polymer and a second polymeric macromolecular species or polymer, wherein the polymeric macromolecular species or polymers are not physically and/or chemically molecularly entangled.

Embodiments of the present disclosure provide an article comprising a substrate and a coating comprising a composite as described in any of the above embodiments. In some embodiments, the substrate comprises an irregular surface. In some embodiments, the thickness of the coating is between about 10 μm and about 1000 μm. In some embodiments, the amount of the coating on the substrate is between about 0.01 g/ ^ft2 and about 25 g/ ^ft2 . In some embodiments, the amount of the first polymeric macromolecular species or polymer in the coating on the substrate is between about 0.001 g/ ^ft2 and about 20 g/ ^ft2 . In some embodiments, the amount of the second polymeric macromolecular species or polymer in the coating on the substrate is between about 0.001 g/ ^ft2 and about 15 g/ ^ft2 . In some embodiments, the substrate comprises a substantially flexible material. In some embodiments, the substrate comprises a leather material or a textile material. In some embodiments, the substrate comprises one or more of collagen, cellulose, and/or lignin.

Embodiments of the present disclosure provide a method for coating a substrate, the method comprising applying a first composition comprising a first polymeric macromolecular species or polymer and a second composition comprising a second polymeric macromolecular species or polymer to the surface of the substrate. In some embodiments, the first composition comprises an unstructured first polymeric macromolecular species or polymer, or a first structure of a first polymeric macromolecular species or polymer. In some embodiments, the first polymeric macromolecular species or polymer comprises a protein component. In some embodiments, the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein and/or whey. In some embodiments, the first polymeric macromolecular species or polymer comprises a biodegradable polymer. In some embodiments, the first polymeric macromolecular species or polymer comprises one or more of polyurethane components. In some embodiments, the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component, a poly(lactic- co -glycolic acid) (PLGA) component, or both. In some embodiments, the second composition comprises an unstructured second polymeric macromolecular species or polymer, or a first structure of a second polymeric macromolecular species or polymer. In some embodiments, the second polymeric macromolecular species or polymer comprises a cellulose and/or cellulose derivative component. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate and microcrystalline cellulose. In some embodiments, the cellulose derivative is ethylcellulose. In some embodiments, the ethoxy content in ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0% or 48.0% to 49.5%. In some embodiments, the degree of substitution of ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5 or 2.5 to 3. In some embodiments, the cellulose derivative comprises a first structure of a cellulose derivative, the crystallinity of which is lower than the second structure of the cellulose derivative, the second structure comprising a crystallinity between about 5% and less than about 100%. In some embodiments, the second composition comprising a second polymeric macromolecular species or polymer further comprises a solvent component. In some embodiments, the solvent component comprises an alcohol and/or an alcohol derivative. In some embodiments, the solvent component comprises one or more of an alcohol, an ether, a ketone, an aldehyde, and/or a ketal. In some embodiments, the solvent component is from about 75% w/w to about 99% w/w of the composition, from about 80% w/w to about 98% w/w of the composition, from about 85% w/w to about 97.5% w/w of the composition, or from about 85% w/w to about 95% w/w of the composition. In some embodiments, the solvent component comprises one or more of methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, pentanol, hexanol, acetone, butanone, methoxypropanol, diisopropylglycerol, 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, 2,2-dimethyl-1,3-dioxolane-4-methanol, or any combination thereof. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer further comprises one or more of a polyethylene glycol (PEG) component, a polypropylene glycol (PPG) component, and/or a polyether component. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer further comprises one or more of a fatty acid or a fatty acid-derived amide and/or one or more of a monoglyceride, a diglyceride, and/or a triglyceride. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer further comprises one or more of a triethylene glycol monomethyl ether component, a diethylene glycol butyl ether component, a diethylene glycol ethyl ether component, a tetradecanedioic acid dimethyl ester component, an erucamide component, and/or a stearic acid glyceryl ester component. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer comprises one or more of an isocyanate component, a polyol component, a blocked isocyanate component, and/or a blocked polyol component. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer comprises a partially polymerized, partially cross-linked, and/or partially cured polyurethane component. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer further comprises a polyurethane prepolymer component. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer further comprises water. In some embodiments, the substrate surface is first coated with a first composition comprising a first polymeric macromolecular species or polymer and then coated with a second composition comprising a second polymeric macromolecular species or polymer. In some embodiments, the method further comprises a drying or partial drying step between the two coating steps. In some embodiments, the first composition comprising the first polymeric macromolecular species or polymer is only partially polymerized, partially dried and/or partially cured before applying the second composition comprising the second polymeric macromolecular species or polymer. In some embodiments, the second composition comprising the second polymeric macromolecular species or polymer is applied at a temperature higher than the glass transition temperature ( _Tg ) of the first polymeric macromolecular species or polymer. In some embodiments, the second composition comprising the second polymeric macromolecular species or polymer is applied at a temperature higher than the glass transition temperature ( _Tg ) of the second polymeric macromolecular species or polymer. In some embodiments, a first composition comprising a first polymeric macromolecular species or polymer is applied one or more times at a rate of about 0.5 mL/ft ² to about 5 mL/ft ^2. In some embodiments, a second composition comprising a second polymeric macromolecular species or polymer is applied one or more times at a rate of about 0.5 mL/ft ² to about 5 mL/ft ² .

Embodiments of the present disclosure provide an article comprising a substrate and a coating, the article being made by a method as described in any of the above embodiments. In some embodiments, a first polymeric macromolecular species or polymer is isotropically distributed on a cross-section of the coating from the substrate-coating interface to the outer surface of the coating. In some embodiments, a first polymeric macromolecular species or polymer is anisotropically distributed on a cross-section of the coating from the substrate-coating interface to the outer surface of the coating. In some embodiments, the concentration of the first polymeric macromolecular species or polymer closer to the substrate-coating interface is higher than the concentration of the first macromolecular species or polymer closer to the outer surface of the coating. In some embodiments, the first polymeric macromolecular species or polymer is substantially undetectable at the outer surface of the coating. In some embodiments, the second polymeric macromolecular species or polymer is isotropically distributed across a cross-section of the coating from the substrate-coating interface to the outer surface of the coating. In some embodiments, the second polymeric macromolecular species or polymer is anisotropically distributed across a cross-section of the coating from the substrate-coating interface to the outer surface of the coating. In some embodiments, the concentration of the second polymeric macromolecular species or polymer closer to the substrate-coating interface is lower than the concentration of the second polymeric macromolecular species or polymer closer to the outer surface of the coating. In some embodiments, the second polymeric macromolecular species or polymer is substantially undetectable at the substrate-coating interface.

Embodiments of the present disclosure provide a composite or a method for making the composite as described in any of the aforementioned embodiments, wherein the composite comprises a matting agent and/or a plasticizer as described herein.

Embodiments of the present disclosure provide an article or a method of making the article as described in any of the aforementioned embodiments, wherein the article comprises a matting agent and/or a plasticizer as described herein.

Embodiments of the present disclosure provide a complex or article, or a method for producing the same, as described in any of the preceding claims, comprising a modified silk fibroin fragment as described herein. Embodiments of the present disclosure provide a plurality of modified silk fibroin fragments, each comprising one or more amino acid residue modifications selected from an asparagine to aspartic acid modification, a glutamine to glutamine modification, and a methionine to methionine oxide modification. Embodiments of the present disclosure provide a plurality of modified silk fibroin fragments comprising one modification. Embodiments of the present disclosure provide a plurality of modified silk fibroin fragments comprising two modifications. Embodiments of the present disclosure provide a plurality of modified silk fibroin fragments comprising three modifications. Examples of the present disclosure provide asparagine to aspartic acid modifications at one or more positions selected from: N23, N28, N108, N118, N136, N186, N200, N204, N240, N248, N68, N70, N77, N5262, N93, N132, N149, N172, N174, N202, N105, N4191. Examples of the present disclosure provide glutamine to glutamine modifications at one or more positions selected from: Q24, Q149, Q202, Q58, Q139, Q275, Q5216, Q255, and Q125. Examples of the present disclosure provide methionine to methionine oxide modifications at position M64. Embodiments of the present disclosure provide various modifications that are independently present in the composition at between about 1% and about 99%. Embodiments of the present disclosure provide a % modification, which is defined as (the number of modified silk fibroin segments having a modification at a specific position divided by the total number of modified silk fibroin segments including that specific position, whether modified or unmodified) x 100.

Embodiments of the present disclosure provide a coating system for coating leather products, comprising a base coating layer and a top coating layer. The base coating layer comprises one or more of a polyurethane dispersion (PUD), a protein component, and a solvent, and the top coating layer comprises one or more of a polyurethane dispersion (PUD), a cellulose derivative, an alcohol solvent, and a glycerol derivative. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate and microcrystalline cellulose. In some embodiments, the cellulose derivative is ethylcellulose. In some embodiments, the ethoxy content of the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%. In some embodiments, the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3. In some embodiments, the second structure of the cellulose derivative comprises less than 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises between about 5% and less than about 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises a degree of crystallinity between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 99%, or between about 90% and about 100%. In some embodiments, the second structure of the cellulose derivative comprises less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81 ... about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 58%, less than about 57%, less than about 56%, less than about 58%, less than about 57%, less than about 56%, less than about 59%, less than about 58%, less than about 57%, less than about 56 ... %, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than In some embodiments, the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein, and/or whey. In some embodiments, the alcohol solvent is methanol, ethanol, acetone, isopropanol, n-butanol, or a combination thereof. In some embodiments, the coating has a thickness between about 10 μm and about 1000 μm. In some embodiments, the amount of the coating on the substrate is between about 0.01 g/ ^ft2 and about 25 g/ ^ft2 .

Embodiments of the present disclosure provide a method for coating a leather product, the method comprising applying a base coating layer and a top coating layer to the surface of the leather, wherein the base coating layer comprises one or more of a polyurethane dispersion (PUD), a protein component, and a solvent, and the top coating layer comprises one or more of a polyurethane dispersion (PUD), a cellulose derivative, an alcohol solvent, and a glycerol derivative. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate and microcrystalline cellulose. In some embodiments, the cellulose derivative is ethylcellulose. In some embodiments, the ethoxy content of the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%. In some embodiments, the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3. In some embodiments, the second structure of the cellulose derivative comprises less than 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises between about 5% and less than about 100% crystallinity. In some embodiments, the second structure of the cellulose derivative comprises a degree of crystallinity between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 99%, or between about 90% and about 100%. In some embodiments, the second structure of the cellulose derivative comprises less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 81%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 80%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81 ... about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 58%, less than about 57%, less than about 56%, less than about 58%, less than about 57%, less than about 56%, less than about 59%, less than about 58%, less than about 57%, less than about 56 ... %, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than In some embodiments, the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein, and/or whey. In some embodiments, the alcohol solvent is methanol, ethanol, acetone, isopropanol, n-butanol, or a combination thereof. In some embodiments, the coating has a thickness between about 10 μm and about 1000 μm. In some embodiments, the amount of the coating on the substrate is between about 0.01 g/ft2 and about 25 g/ft2.

In some embodiments, the present disclosure provides a composition comprising a coating comprising two components. In some embodiments, the second component is impregnated onto the first component. In some embodiments, the second component undergoes a phase change (such as, but not limited to, Tg, polymerization, etc.). The first coating described herein may include, but is not limited to, a polymer disclosed herein or any protein, such as biodegradable polyurethane, silk protein, collagen, casein, elastin, etc. The second coating described herein may include, but is not limited to, a cellulose derivative disclosed herein. The first coating and the second coating should not be limited in this order, as any coating disclosed herein may be interchangeable with any other coating disclosed herein. While ethyl cellulose can typically be brittle and break, in some embodiments, the present disclosure provides flexible ethyl cellulose coatings. The present disclosure provides protective coatings for coating any surface, including, but not limited to, leather, fabric, wood, and food (fruits, vegetables, etc.). In some embodiments, the coatings disclosed herein are made from two or more films (which can begin with a single film made from two polymers) applied as a monolayer to a substrate. As disclosed herein, the composites and/or coatings disclosed herein can be based on, but not limited to, molecular entanglement, such that the EC does not contain a crosslinking agent. In some embodiments, all layers are held together by molecular interactions. In some embodiments, all molecular interactions result in solidification, coagulation, or polymerization. In some embodiments, the two layers undergo molecular interaction, thereby solidifying the film and forming a larger polymeric structure. In some embodiments, the outer layer described herein comprises 1% to 100% EC on the surface. In some embodiments, the first layer (when applied against the surface to be coated): participates in molecular entanglement, such as adhesion of the first and second layers; the first layer can adhere to uneven surfaces; the first layer is: thermoplastic, self-assembles, and is soluble in the solvent used for the second layer; the first layer polymerizes by crosslinking and self-assembly. In some embodiments, the first layer is resolvable and solidifiable. In some embodiments, a polymer or protein, such as, but not limited to, silk protein, plays a role in the first layer. In some embodiments, the second layer (deposited on top of the first layer and outer layer) is made of ethylcellulose (EC) or a biomaterial or polymer in a molecular dispersion; in some embodiments, this layer contains about 1-5 gr/L of EC by volume in a solvent. In some embodiments, this layer can deliver dyes, silk, or other molecules to modify optical, tactile, and mechanical properties. In some embodiments, the EC is a protective barrier that enhances the performance and characteristics of the first layer. In some embodiments, the EC is mechanically resilient and enhances water resistance properties. In some embodiments, the EC can adhere to a dynamic first layer substrate. In some embodiments, the EC can adhere to uneven first layer surfaces. In some embodiments, the majority of the EC faces outward to the external environment/forces. In some embodiments, proteins or polymers, such as but not limited to silk, function in the second layer.

Silk-coated leather products and methods of making the same have been described in WO 2020/018821 and WO 2021/146654, each of which is incorporated herein by reference in its entirety.

Leather is a material produced by treating the skin, which has been removed from an animal, with a series of physical, mechanical, and chemical processes, followed by tanning. Leather is composed of interwoven collagen fiber bundles, with trace amounts of elastic and reticular fibers, of which the collagen fiber content ranges between 95% and 98%. The natural interwoven structure of collagen fibers in natural leather is characterized by thicker fiber bundles sometimes splitting into several finer fiber bundles, with the resulting finer fiber bundles sometimes joining other fiber bundles to form another larger fiber bundle.

In its natural state, leather is a non-woven material in which the protofibrils of the fibers have grown together. The fibroin and collagen fibers in leather are naturally occurring proteins composed of 22 protein-forming amino acids. Fibroin has a high affinity for leather fibers (collagen) due to the presence of hydrophilic amino acid residues in fibroin (e.g., due to the physical attachment of fibroin fragments to leather fibers through hydrogen bonds), such as the -OH group from serine, the guanidine group from arginine, the free amine group from lysine, and the -COOH groups from aspartic acid and glutamine.

Methods for coating leather include, for example, the following: ● The leather blank may first be treated with a putty and/or an abrasive to prepare the surface for application of the primer. Putty is typically used for leather with scars and deep defects and can be applied by rollers. Abrasives are typically used to polish and/or split the leather and can be applied via a roller coater or spray coater. ● Separately, a suitably textured release paper with a negative image of a printed design engraved thereon is applied with the primer solution and dried. The two substrates (the treated leather and the coated release paper) are then stacked together with the coated sides facing each other. The stacked substrates are then laminated using high pressure and high temperature. After lamination, the release paper is removed, leaving the printed design on the semi-finished leather. ●    To finish the leather, a top coat is applied using a spray or roll coater, dried, and ironed.

In some embodiments, the fibroin-based protein fragments and solutions described herein can be used as color enhancers for leather or leather products. In some embodiments, the present disclosure provides treated leather or leather products that exhibit good dyeability, excellent color fastness, and enhanced color saturation.

The treatment of leather and leather products with silk fibroin-based protein fragments and solutions enhances the quality and aesthetic properties of natural leather using non-toxic, sustainable, and natural silk-based compositions. The silk treatment process disclosed herein promotes advancements in leather products while respecting traditions and craftsmanship without disrupting the leather tanning and creation process. SPF Definition and Properties

As used herein, "silk protein fragment" (SPF) includes, but is not limited to, one or more of the following: a "fibroin fragment" as defined herein; a "recombinant silk fragment" as defined herein; a "spider silk fragment" as defined herein; a "fibroin-like protein fragment" as defined herein; a "chemically modified silk fragment" as defined herein; and/or a "sericulture or sericulture fragment" as defined herein. The SPF can have any molecular weight value or range described herein, and any polydispersity value or range described herein. As used herein, in some embodiments, the term "silk protein fragment" also refers to a silk protein comprising or consisting of at least two identical repeat units, each of which is independently selected from a naturally occurring silk polypeptide or a variant thereof, an amino acid sequence of a naturally occurring silk polypeptide, or a combination of the two. SPM Molecular Weight and Polydispersity

In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 to about 70 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 70 to about 75 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 75 to about 80 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 80 to about 85 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 85 to about 90 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 90 to about 95 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 95 to about 100 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 to about 170 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 170 to about 175 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 175 to about 180 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 180 to about 185 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 185 to about 190 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 190 to about 195 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 195 to about 200 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 200 to about 205 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 205 to about 210 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 210 to about 215 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 215 to about 220 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 220 to about 225 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 225 to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 to about 255 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 to about 260 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 to about 270 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 270 to about 275 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 275 to about 280 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 280 to about 285 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 285 to about 290 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 to about 300 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 to about 310 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 to about 330 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 330 to about 335 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 335 to about 340 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 340 to about 345 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 345 to about 350 kDa.

In some embodiments, the compositions of the present disclosure include an SPF composition selected from compositions #1001 to #2450 having a weight average molecular weight selected from about 1 kDa to about 145 kDa and a polydispersity selected from 1 to about 5 (including but not limited to a polydispersity of 1), 1 to about 1.5 (including but not limited to a polydispersity of 1), about 1.5 to about 2, about 1.5 to about 3, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, and about 4.5 to about 5:

In some embodiments, as will be understood by those skilled in the art, the molecular weights described herein can be converted to an approximate number of amino acids contained in the corresponding SPF. For example, the average weight of amino acids can be approximately 110 daltons (i.e., 110 g/mol). Therefore, in some embodiments, dividing the molecular weight of a linear protein by 110 daltons can be used to approximate the number of amino acid residues contained therein.

In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from 1 to about 5.0, including but not limited to a polydispersity of 1. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from 1 to about 1.5, including but not limited to a polydispersity of 1. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 1.5 to about 2.0. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 2.0 to about 2.5. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 2.5 to about 3.0. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 3.0 to about 3.5. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 3.5 to about 4.0. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF of the composition of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

In one embodiment, the polydispersity of SPF in the composition of the present disclosure is 1. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.1. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.2. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.3. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.4. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.5. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.6. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.7. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.8. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 1.9. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.0. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.1. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.2. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.3. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.4. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.5. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.6. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.7. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.8. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 2.9. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.0. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.1. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.2. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.3. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.4. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.5. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.6. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.7. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.8. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 3.9. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.0. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.1. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.2. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.3. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.4. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.5. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.6. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.7. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.8. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 4.9. In one embodiment, the polydispersity of SPF in the composition of the present disclosure is about 5.0.

In some embodiments, in compositions described herein having a combination of low, medium, and/or high molecular weight SPFs, such low, medium, and/or high molecular weight SPFs may have the same or different polydispersities. Fibroin Fragments

Methods for producing fibroin or fibroin fragments and their applications in various fields are known and described, for example, in U.S. Patents Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, 10,166,177, 10,287,728, and 10,301,768, all of which are incorporated herein in their entireties. Raw silk from silkworm ( Bombyx mori ) is composed of two main proteins: fibroin (approximately 75%) and sericin (approximately 25%). Fibroin is a fibrous protein with a semicrystalline structure that provides rigidity and strength. As used herein, the term "fibroin" refers to the fibers of silkworms with a weight-average molecular weight of approximately 370,000 Da. Thick silk fibers are composed of double strands of fibroin. The adhesive that holds these double strands of fibers together is silk glue. Fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Fibroin is a high-molecular-weight amphiphilic polymer with large hydrophobic domains that constitute the majority of the polymer. The hydrophobic regions are interspersed with small hydrophilic spacers, and the N- and C-termini of the chain are also highly hydrophilic. The hydrophobic domains of the H chain contain repeating hexapeptide sequences of Gly-Ala-Gly-Ala-Gly-Ser and repeating dipeptide sequences of Gly-Ala/Ser/Tyr, which form stable antiparallel platelets. The L chain does not have a repeating amino acid sequence, making it more hydrophilic and relatively flexible. The alternating arrangement of hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) segments in the fibroin molecule enables self-assembly.

Provided herein are methods for generating pure and highly scalable fibroin-protein fragment mixture solutions that can be used in a variety of applications across multiple industries. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to the fragmentation of any SPF described herein, including but not limited to recombinant fibroin, as well as the fragmentation of fibroin-like or fibroin-like proteins.

As used herein, the term "fibroin" includes silkworm silk fibroin and insect or spider silk proteins. In one embodiment, the fibroin is obtained from silkworms. Raw silk from silkworms is composed of two main proteins: fibroin (approximately 75%) and sericin (approximately 25%). Fibroin is a fibrous protein with a semicrystalline structure that provides rigidity and strength. As used herein, the term "fibroin" refers to silkworm fibers with a weight-average molecular weight of approximately 370,000 Da. Converting these insoluble fibroin protofibrils into water-soluble fibroin fragments requires the addition of concentrated neutral salts (e.g., 8-10 M lithium bromide), which disrupt the intermolecular and intramolecular ionic and hydrogen bonds, otherwise rendering the fibroin insoluble in water. Methods for producing fibroin fragments and/or compositions thereof are known and described, for example, in U.S. Patents Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

Raw silk coils from silkworms are cut into pieces. The coil pieces are treated in an aqueous solution of _Na₂CO₃ at approximately 100° _C for approximately 60 minutes to remove the silk glue (degumming). The volume of water used is approximately equal to 0.4 times the weight of the raw silk, and the _amount of _Na₂CO₃ is approximately 0.848 times the weight of the raw silk coil pieces. The resulting degummed silk coil pieces are rinsed three times with deionized water at approximately 60°C (20 minutes each). The volume of rinse water in each cycle is 0.2 L x the weight of the raw silk coil pieces. Excess water from the degummed silk coil pieces is removed. After the deionized water washing step, the wet degummed silk coil fragments are dried at room temperature. The degummed silk coil fragments are mixed with a LiBr solution and the mixture is heated to about 100°C. The heated mixture is placed in a drying oven and heated at about 100°C for about 60 minutes to achieve complete dissolution of the native silk protein. The resulting silk fibrin solution is filtered and dialyzed against deionized water using a tangential flow filtration (TFF) and a 10 kDa membrane for 72 hours. The concentration of the resulting silk fibrin aqueous solution is about 8.5% by weight. Subsequently, the 8.5% silk solution is diluted with water to produce a 1.0% w/v silk solution. The pure silk solution can then be further concentrated using TFF to a concentration of 20.0% w/w silk in water.

Dialysis of silk using a series of water changes is a manual and time-intensive process that can be accelerated by varying certain parameters, such as diluting the silk solution prior to dialysis. The dialysis process can be scaled up using semi-automated equipment, such as tangential flow filtration systems.

In some embodiments, silk solutions were prepared under various preparation conditions, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3 M LiBr was prepared and allowed to stand at room temperature for at least 30 minutes. 5 mL of the LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples from each group were removed at 4, 6, 8, 12, 24, 168, and 192 hours.

In some embodiments, silk solutions were prepared under various preparation conditions, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3 M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of the hot LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples from each group were removed after 1, 4, and 6 hours.

In some embodiments, silk solutions are prepared under various preparation conditions, such as using four different silk extraction combinations: 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3 M LiBr solution is heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of hot LiBr solution is added to 1.25 g of silk and placed in an oven at the same temperature as the LiBr. Samples from each group are removed at 1, 4, and 6 hours. 1 mL of each sample is added to 7.5 mL of 9.3 M LiBr and refrigerated for viscosity testing.

In some embodiments, SPF is obtained by dissolving unwashed, partially washed, or washed raw silk with a neutral lithium bromide salt. The raw silk is treated at a selected temperature and other conditions to remove any silk glue and achieve the desired weight-average molecular weight ( _MW ) and polydispersity (PD) of the fragment mixture. Depending on the intended application, the selection of process parameters can be varied to achieve different final silk protein fragment characteristics. The resulting final fragment solution is silk fibroin fragments and water, which has process contaminants ranging from parts per million (ppm) to undetectable levels, i.e., levels acceptable to the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further varied depending on the desired application and performance requirements.

FIG1 is a flow chart showing various embodiments of the present disclosure for producing pure silk fibroin fragments (SPF). It should be understood that not all steps shown are necessary to produce all silk solutions disclosed herein. As shown in FIG1 , step A, coils (heat-treated or not), silk fibers, silk powder, spider silk, or recombinant spider silk can be used as the silk source. If starting with raw silk coils from silkworms, the coils can be cut into small pieces, for example, pieces of approximately equal size, step B1. The raw silk is then extracted and rinsed to remove any silk glue, step C1a. This produces raw silk that is substantially free of silk glue. In one embodiment, water is heated to a temperature between 84°C and 100°C (ideally boiling), and _Na₂CO₃ ( _sodium carbonate) is then added to the boiling water until _{the Na₂CO₃} is completely dissolved. Raw silk is added to the boiling water/ _Na₂CO₃ (100° _C ) and immersed for approximately 15-90 minutes, with longer boiling times resulting in smaller silk protein fragments. In one embodiment, the volume of water is equal to approximately 0.4× the weight of the raw silk, and _the volume of _Na₂CO₃ is equal to approximately 0.848× the weight of the raw silk. In another embodiment, the volume of water is equal to 0.1× the weight of the raw silk, and the volume _{of Na₂CO₃} is maintained at 2.12 g/L.

Subsequently, the _{aqueous Na₂CO₃} solution is drained, and excess water/ _Na₂CO₃ is removed from the fibrous protein fibers (e.g., by manually circulating the fibrous protein extract, using a rotary circulation machine, etc.). The resulting fibrous protein extract is rinsed with warm to hot water to remove any remaining adsorbed silk glue or contaminants, typically at a temperature ranging from about 40° _C to about 80°C, changing the volume of water at least once (repeated as needed). The resulting fibrous protein extract is fibrous protein with silk glue substantially removed. In one embodiment, the resulting fibrous protein extract is rinsed with water at a temperature of about 60°C. In one embodiment, the volume of the rinse water in each cycle is equal to 0.1 L to 0.2 L x the weight of the raw silk. It may be advantageous to agitate, rotate or circulate the rinse water to maximize the rinsing effect. After rinsing, excess water is removed from the extracted silk fibroin fibers (for example, by hand or using a machine to loop out the silk fibroin extract). Alternatively, methods known to those skilled in the art (such as pressure, temperature or other reagents or a combination thereof) can be used for the purpose of silk silk extraction. Alternatively, the silk glands (100% silk protein free of silk) can be removed directly from the silkworm. This will produce liquid silk protein free of silk without any change in the protein structure.

The extracted fibroin fibers are then completely dried. Once dried, the extracted fibroin is dissolved in a solvent that is added to the fibroin at a temperature between ambient temperature and boiling temperature, step C1b. In one embodiment, the solvent is a lithium bromide (LiBr) solution (LiBr has a boiling point of 140°C). Alternatively, the extracted fibroin fibers are not dried, but rather wetted and placed in the solvent; the solvent concentration can then be varied to achieve a concentration similar to that when the dried silk is added to the solvent. The final concentration of the LiBr solvent can range from 0.1 M to 9.3 M. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature, as well as the concentration of the dissolving solvent. Other solvents may be used, including but not limited to phosphate phosphoric acid, calcium nitrate, calcium chloride solutions, or other concentrated inorganic salt solutions. To ensure complete dissolution, the fibers should be completely immersed in the heated solvent solution and then maintained at a temperature in the range of about 60°C to about 140°C for 1-168 hours. In one embodiment, the fibers should be completely immersed in the solvent solution and then placed in a drying oven at a temperature of about 100°C for about 1 hour.

The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an impact on the time required to completely dissolve the silk fibroin and the resulting molecular weight and polydispersity of the final SPF mixture solution. In one embodiment, the silk solvent solution concentration is less than or equal to 20% w/v. In addition, agitation during introduction or dissolution can be used to promote dissolution at different temperatures and concentrations. The temperature of the LiBr solution will provide control over the molecular weight and polydispersity of the resulting silk protein fragment mixture. In one embodiment, higher temperatures will dissolve the silk more quickly, thereby providing enhanced process scalability and large-scale production of silk solutions. In one embodiment, using a LiBr solution heated to a temperature of 80°C to 140°C reduces the time required to achieve complete dissolution in an oven. Changing the time and temperature of the dissolution solvent at 60°C or above will change and control the MW and polydispersity of the SPF mixture solution formed from native silk fibroin of original molecular weight.

Alternatively, the entire coil can be placed directly in a solvent such as LiBr, bypassing extraction (step B2). This requires subsequent filtration of the silkworm particles from the silk and solvent solution, and removal of the silkworm using methods known in the art for separating hydrophobic and hydrophilic proteins, such as column separation and/or chromatography, ion exchange, chemical precipitation using salt and/or pH, and/or enzymatic digestion and filtration or extraction, all of which are common examples of, but not limited to, standard protein separation methods (step C2). Alternatively, the unheated coil, from which the silkworms have been removed, can be placed in a solvent such as LiBr, bypassing extraction. The above method can be used for silk silk separation, with the advantage that the unheat-treated silk cocoons will contain significantly less silk debris.

Dialysis can be used to remove the dissolving solvent from the resulting solubilized silk fibroin fragment solution by dialyzing the solution against a certain volume of water, step E1. Pre-filtration prior to dialysis helps remove any debris (i.e., residual matter) from the silk and LiBr solution, step D. In one example, a 0.1% to 1.0% silk-LiBr solution is filtered using a 3 μm or 5 μm filter at a flow rate of 200-300 mL/min prior to dialysis and may be concentrated if necessary. As described above, the methods disclosed herein use time and/or temperature to reduce the concentration from 9.3 M LiBr to a range of 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering the creation of a scalable process. Alternatively, without using additional time or temperature, the 9.3 M LiBr-filament protein fragment solution can be diluted with water to facilitate debris filtration and dialysis. The result of filtration at the desired time and temperature is a translucent, particle-free, room temperature storage stable filament protein fragment-LiBr solution of known MW and polydispersity. It is advantageous to regularly replace the dialysis water until the solvent is removed (e.g., replace the water after 1 hour, 4 hours, and then every 12 hours, for a total of 6 water changes). The total amount of water volume change can vary based on the resulting concentration of the solvent used for silk protein solubilization and fragmentation. After dialysis, the final silk solution can be further filtered to remove any remaining debris (ie, silk residues).

Alternatively, tangential flow filtration (TFF), a rapid and efficient method for separating and purifying biomolecules, can be used to remove the solvent from the resulting dissolved silk fibroin solution, step E2. TFF provides a highly pure aqueous solution of silk fibroin fragments and enables the scalability of the method to produce large quantities of solution in a controlled and reproducible manner. The silk and LiBr solutions can be diluted prior to TFF (from 20% to 0.1% silk in water or LiBr). Prefiltration prior to TFF treatment as described above can maintain filter efficiency and potentially avoid the formation of a silk gel boundary layer on the filter surface due to the presence of debris particles. Prefiltration prior to TFF also helps remove any remaining debris (i.e., silk residue) from the silk and LiBr solution, which could cause spontaneous or prolonged gelation of the resulting water-only solution, step D. TFF (recirculating or single-pass) can be used to produce water-silk protein fragment solutions with concentrations ranging from 0.1% silk to 30.0% silk (more preferably 0.1%-6.0% silk). Different cutoff TFF membranes may be required based on the desired concentration, molecular weight, and polydispersity of the silk protein fragment mixture in the solution. For example, membranes in the 1-100 kDa range may be required for silk solutions of varying molecular weights, produced by varying the length of the extraction boiling time or the time and temperature in the dissolution solvent (e.g., LiBr). In one embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution and produce the final desired silk to water ratio. Similarly, TFF single pass, TFF and other methods known in the art, such as falling film evaporators, can be used to concentrate the solution after removing the dissolving solvent (e.g., LiBr) (to obtain the desired concentration range of 0.1% to 30% silk). This can be used as an alternative to the standard HFIP concentration method known in the art to produce a water-based solution. Larger pore membranes can also be used to filter out small silk protein fragments and produce solutions with and/or without tighter polydispersity values of higher molecular weight silk.

The analysis for LiBr and _Na2CO3 detection can be performed using an HPLC system equipped with an evaporative _light scattering detector (ELSD). Calculations are performed by linear regression of the resulting peak area of the analyte plotted against concentration. More than one sample of the various formulations disclosed herein is used for sample preparation and analysis. Typically, four samples of different formulations are weighed directly into a 10 mL volumetric flask. The sample is suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and kept at 2-8°C for 2 hours with occasional shaking to extract the analyte from the membrane. After 2 hours, the solution is diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask is transferred to an HPLC vial and injected into the HPLC-ELSD system for estimation of sodium carbonate and lithium bromide.

The developed analytical method for the quantification of _Na₂CO₃ and LiBr in _silk protein formulations was found to be linear over the range of 10-165 μg/mL. For sodium carbonate and lithium bromide, the RSDs for injection precision were 2% and 1% by area, and 0.38% and 0.19% by retention time, respectively. This analytical method can be used for the quantitative determination of sodium carbonate and lithium bromide in silk protein formulations.

FIG2 is a flow chart illustrating various parameters that can be modified during the extraction and solubilization steps of a process for producing a silk protein fragment solution disclosed herein. Depending on the intended application, the selected process parameters can be varied to achieve different final solution characteristics, such as molecular weight and polydispersity. It should be understood that not all steps depicted are required to produce all silk solutions disclosed herein.

In one embodiment, a silk protein fragment solution useful in various applications is prepared according to the following steps: forming silk cocoon fragments from silkworms; extracting the fragments in an _{aqueous Na2CO3} solution at about 100°C for about 60 minutes, wherein the volume of water is equal to about 0.4 times the weight of the raw silk and _the amount of _Na2CO3 is about 0.848 times the weight of the fragments to form a silk fiber protein extract; rinsing the silk fiber protein extract three times in a certain volume of rinse water at about 60°C, each rinse for about 20 minutes, wherein the rinse water for each cycle is equal to about 0.2 L x weight of fragments; removing excess water from the silk fibroin extract; drying the silk fibroin extract; dissolving the dried silk fibroin extract in a LiBr solution, wherein the LiBr solution is first heated to approximately 100°C to produce a silk and LiBr solution and maintained; placing the silk and LiBr solution in a drying oven at approximately 100°C for approximately 60 minutes to achieve complete dissolution of the native silk protein structure and further fragmentation into a mixture with the desired molecular weight and polydispersity; filtering the solution to remove any remaining silk debris; diluting the solution with water to produce a 1.0 wt% silk solution; and removing the solvent from the solution using tangential flow filtration (TFF). In one embodiment, a 10 kDa membrane is used to purify the silk solution and produce the final desired silk to water ratio. The silk solution can then be further concentrated using TFF to a concentration of 2.0 wt% silk in water.

Without wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution when added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent and silk solutions with varying viscosities, homogeneities, and colors. Also without wishing to be bound by any particular theory, increasing the extraction temperature, extending the extraction time, using higher temperature LiBr solutions over time during immersion and while dissolving the silk, and increasing the time at temperature (e.g., in an oven as described herein, or with an alternative heat source) all resulted in less viscous and more homogeneous solvents and silk solutions.

The extraction step can be accomplished in a larger vessel, such as an industrial washer, in which a temperature of 60°C to 100°C or between 60°C and 100°C can be maintained. The rinsing step can also be accomplished in an industrial washer, thereby eliminating manual rinse cycles. The dissolution of the silk in the LiBr solution can occur in a vessel other than a convection oven, such as a stirred tank reactor. Dialysis of the silk through a series of water changes is a manual and time-intensive process that can be accelerated by varying certain parameters, such as diluting the silk solution prior to dialysis. The dialysis process can be scaled up for manufacturing by using semi-automated equipment, such as a tangential flow filtration system.

Varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution when added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent and silk solutions with varying viscosities, homogeneities, and colors. Increasing the extraction temperature, extending the extraction time, using higher temperature LiBr solutions during immersion and over time while dissolving the silk, and increasing the time at temperature (e.g., in an oven as described herein, or with an alternative heat source) all resulted in less viscous and more homogeneous solvent and silk solutions. While nearly all parameters produced viable silk solutions, methods that allowed complete dissolution to be achieved in less than 4 to 6 hours were preferred for process scalability.

In one embodiment, a solution of silk fibroin fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa is prepared according to the following steps: degumming a silk source by adding the silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes; removing the silk glue from the solution to produce a silk fibroin extract containing an undetectable level of silk glue; draining the solution from the silk fibroin extract; and separating the silk fibroin extract from the silk fibroin extract. The method further comprises dissolving the fibroin extract in a lithium bromide solution, wherein the initial temperature of the solution is in the range of about 60° C. to about 140° C. when the fibroin extract is placed in the lithium bromide solution; maintaining the fibroin-lithium bromide solution in an oven at a temperature of about 140° C. for a period of up to 1 hour; removing the lithium bromide from the fibroin extract; and producing an aqueous solution of fibroin fragments, the aqueous solution comprising fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the fibroin extract prior to the solubilization step. The aqueous solution of fibroin fragments may contain less than 300 ppm of lithium bromide residues as measured by high-performance liquid chromatography (HPLC) with lithium bromide analysis. The aqueous solution of fibroin fragments may contain less than 100 ppm of sodium carbonate residues as measured by high-performance liquid chromatography (HPLC) with sodium carbonate analysis. The aqueous solution of fibroin fragments may be freeze-dried. In some embodiments, the solution of fibroin fragments may be further processed into various forms, including gels, powders, and nanofibers.

In one embodiment, a solution of silk fibroin fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa is prepared according to the following steps: adding a silk source to a boiling (100° C.) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes, thereby causing degumming; removing the silk glue from the solution to produce a silk fibroin extract containing an undetectable level of silk glue; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in lithium bromide; The method comprises the steps of: placing a fibrous protein extract in a lithium bromide solution at an initial temperature ranging from about 80° C. to about 140° C.; maintaining the fibrous protein-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for a period of up to 1 hour; removing lithium bromide from the fibrous protein extract; and producing an aqueous solution of fibrous protein fragments, wherein the aqueous solution of fibrous protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of the silk fibroin fragments contains about 10 ppm to about 100 ppm of sodium carbonate residues, wherein the aqueous solution of the silk fibroin fragments comprises fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the solubilization step. The aqueous solution of the silk fibroin fragments may contain less than 300 ppm of lithium bromide residues as measured using high performance liquid chromatography (HPLC) lithium bromide analysis. The aqueous solution of the silk fibroin fragments may contain less than 100 ppm of sodium carbonate residues as measured using high performance liquid chromatography (HPLC) sodium carbonate analysis.

In some embodiments, a method for preparing an aqueous solution of silk fibroin fragments having an average weight average molecular weight selected from about 6 kDa to about 17 kDa comprises the steps of: degumming a silk source by adding the silk source to a boiling (100° C.) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes; removing the silk glue from the solution to produce a silk fibroin extract comprising an undetectable level of silk glue; draining the solution from the silk fibroin extract; and removing the silk fibroin from the solution. The fibroin extract is dissolved in a lithium bromide solution, wherein the initial temperature of the solution is in the range of about 60° C. to about 140° C. when the fibroin extract is placed in the lithium bromide solution; the fibroin-lithium bromide solution is maintained in an oven at a temperature of about 140° C. for a period of up to 1 hour; the lithium bromide is removed from the fibroin extract; and an aqueous solution of fibroin fragments is produced, the aqueous solution comprising fragments having an average weight-average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the fibroin extract prior to the solubilization step. The aqueous solution of the pure filament protein fragments may contain less than 300 ppm of lithium bromide residues as measured using high-performance liquid chromatography (HPLC) lithium bromide analysis. The aqueous solution of the pure filament protein fragments may contain less than 100 ppm of sodium carbonate residues as measured using high-performance liquid chromatography (HPLC) sodium carbonate analysis. The method may further include adding a therapeutic agent to the aqueous solution of the pure filament protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of the pure filament protein fragments. The method may further include adding a vitamin to the aqueous solution of the pure filament protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of the pure filament protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to an aqueous solution of pure fibrous protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further comprise adding hyaluronic acid or a salt thereof at a concentration of about 0.5% to about 10.0% to an aqueous solution of pure fibrous protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A membrane may be made from an aqueous solution of pure fibrous protein fragments produced by this method. The membrane may comprise about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The membrane may have a water content ranging from about 2.0% to about 20.0% by weight. The membrane may comprise from about 30.0% to about 99.5% by weight of the pure silk fibroin fragments. A gel may be prepared from an aqueous solution of the pure silk fibroin fragments produced by this method. The gel may comprise from about 0.5% to about 20.0% by weight of vitamin C or its derivatives. The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

In some embodiments, a method for preparing an aqueous solution of silk fibroin fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa comprises the steps of: adding a silk source to a boiling (100° C.) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes, thereby causing degumming; removing the silk glue from the solution to produce a silk fibroin extract containing an undetectable level of silk glue; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in brominated water; The invention relates to a method for preparing a fibrous protein extract and a lithium bromide solution, wherein the initial temperature of the solution is in the range of about 80° C. to about 140° C. when the fibrous protein extract is placed in the lithium bromide solution; maintaining the fibrous protein-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for a period of up to 1 hour; removing lithium bromide from the fibrous protein extract; and producing an aqueous solution of pure fibrous protein fragments, wherein the aqueous solution of pure fibrous protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of the silk fibroin fragments contains about 10 ppm to about 100 ppm of sodium carbonate residues, wherein the aqueous solution of the pure silk fibroin fragments comprises fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the solubilization step. The aqueous solution of the pure silk fibroin fragments may contain less than 300 ppm of lithium bromide residues as measured using high performance liquid chromatography (HPLC) lithium bromide analysis. The aqueous solution of pure fibrous protein fragments may contain less than 100 ppm of sodium carbonate residues as measured using high performance liquid chromatography (HPLC) sodium carbonate analysis. The method may further include adding a therapeutic agent to the aqueous solution of pure fibrous protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure fibrous protein fragments. The method may further include adding a vitamin to the aqueous solution of pure fibrous protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure fibrous protein fragments may be lyophilized. The method may further include adding an α-hydroxy acid to the aqueous solution of pure fibrous protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further comprise adding hyaluronic acid or a salt thereof at a concentration of about 0.5% to about 10.0% to an aqueous solution of pure silk fibroin fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A membrane may be made from the aqueous solution of pure silk fibroin fragments produced by this method. The membrane may comprise about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The membrane may have a water content ranging from about 2.0% to about 20.0% by weight. The membrane may comprise about 30.0% to about 99.5% by weight of pure silk fibroin fragments. A gel can be made from an aqueous solution of pure silk fibroin fragments produced by this method. The gel can contain from about 0.5% to about 20.0% by weight of vitamin C or its derivatives. The gel can have a silk content of at least 2% and a vitamin content of at least 20%.

In one embodiment, a solution of silk fibroin fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes, thereby causing degumming; removing the silk glue from the solution to produce a silk fibroin extract containing an undetectable level of silk glue; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution; , when the fibrous protein extract is placed in the lithium bromide solution, the initial temperature of the solution is in the range of about 80° C. to about 140° C.; maintaining the fibrous protein-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for a period of up to 1 hour; removing lithium bromide from the fibrous protein extract; and producing an aqueous solution of fibrous protein fragments, wherein the aqueous solution of fibrous protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residues, about 10 ppm to about 100 ppm of sodium carbonate residues, and fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the fibrous protein extract prior to the solubilization step. The aqueous solution of the fibrous protein fragments may comprise less than 300 ppm of lithium bromide residues as measured using high performance liquid chromatography (HPLC) lithium bromide analysis. The aqueous solution of the fibrous protein fragments may comprise less than 100 ppm of sodium carbonate residues as measured using high performance liquid chromatography (HPLC) sodium carbonate analysis. In some embodiments, the method may further comprise adding an active agent (e.g., a therapeutic agent) to the aqueous solution of the purified fibrous protein fragments. The method may further comprise adding an active agent selected from one of an antioxidant or an enzyme to the aqueous solution of the purified fibrous protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure fibrous protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure fibrous protein fragments may be freeze-dried. The method may further comprise adding an α-hydroxy acid to the aqueous solution of pure fibrous protein fragments. The α-hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further comprise adding hyaluronic acid or a salt thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure fibrous protein fragments. A membrane may be made from the aqueous solution of pure fibrous protein fragments produced by this method. The membrane may comprise about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The membrane may have a water content ranging from about 2.0% to about 20.0% by weight. The membrane may comprise about 30.0% to about 99.5% by weight of pure silk fibroin fragments. Gels may be prepared from aqueous solutions of pure silk fibroin fragments produced by this method. The gel may comprise about 0.5% to about 20.0% by weight of vitamin C or its derivatives. The gel may have a silk content of at least 2% by weight and a vitamin content of at least 20% by weight.

The molecular weight of the silk protein fragments can be controlled based on specific parameters used during the extraction step, including extraction time and temperature; specific parameters used during the dissolution step, including the LiBr temperature when the silk is immersed in lithium bromide and the time the solution is maintained at a specific temperature; and specific parameters used during the filtration step. By controlling the process parameters using the disclosed method, a silk fibroin fragment solution with a polydispersity equal to or less than 2.5 can be produced, which has a variety of different molecular weights selected from 5 kDa to 200 kDa or 10 kDa to 80 kDa. By varying the process parameters to obtain silk solutions with different molecular weights, a range of fragment mixture final products with a desired polydispersity equal to or less than 2.5 can be targeted based on the desired performance requirements. For example, a higher molecular weight silk film containing an ophthalmic drug can have a controlled, slow release rate compared to a lower molecular weight film, making it an ideal choice for a delivery vehicle in eye care products. In addition, a silk fibroin fragment solution with a polydispersity greater than 2.5 can be obtained. In addition, two solutions with different average molecular weights and polydispersities can be mixed to produce a combined solution. Alternatively, liquid silk glands removed directly from silkworms (100% silk protein without silk glue) can be used in combination with any of the silk fibroin fragment solutions disclosed herein. The molecular weight of the pure silk fibroin fragment composition was determined using high pressure liquid chromatography (HPLC) with a refractive index detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC/SEC software version 3.3 (Agilent).

Differences in processing parameters can produce regenerated silk fiber proteins with varying molecular weights and peptide chain size distributions (polydispersity, PD). This, in turn, affects the properties of the regenerated silk fiber proteins, including mechanical strength and water solubility.

Parameters were varied during the processing of raw silk into a silk solution. These parameters affected the MW of the resulting silk solution. The manipulated parameters included (i) extraction time and temperature, (ii) LiBr temperature, (iii) dissolution oven temperature, and (iv) dissolution time. Experiments were conducted to determine the effects of varying extraction time. Tables A and B summarize the results. Here is an overview: - A 30 minute silk silk extraction time produced higher molecular weights than a 60 minute silk silk extraction time - The molecular weight decreased with time in the oven - 140°C LiBr and an oven resulted in a lower confidence limit of less than 9500 Da molecular weight - The 30 minute extraction at the 1 hour and 4 hour time points had undigested silk - The 30 minute extraction at the 1 hour time point produced significantly higher molecular weights with a lower confidence limit of 35,000 Da - The upper limit of the confidence limit achieved a molecular weight range of 18,000 to 216,000 Da (critical to provide a solution with the specified upper limit).

Experiments were conducted to determine the effect of varying the extraction temperature. Table G summarizes the results. Here is a summary: - Silk silk extracted at 90°C produced a higher MW than silk silk extracted at 100°C - Both 90°C and 100°C showed a decrease in MW over time in the oven.

Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) when added to silk. Table HI summarizes the results. The following is a summary: - No effect on molecular weight or confidence intervals (all CI ~10500-6500 Da) - The study showed that when LiBr was added and dissolution began, the temperature at which the LiBr-silk dissolved quickly dropped below the original LiBr temperature. This is due to the fact that most of the mass is in the silk at room temperature.

Experiments were conducted to determine the effect of v oven/dissolution temperature. Table JN summarizes the results. The following is a summary: - The effect of oven temperature on silk extracted for 60 minutes was less than that on silk extracted for 30 minutes. Without wishing to be bound by theory, it is believed that the 30 minute silk degraded less during extraction and therefore the oven temperature had a greater effect on the larger MW, less degraded portion of the silk. - For both the 60°C and 140°C ovens, the 30 minute extracted silk showed a very significant effect on the lower MW at the higher oven temperature, while the 60 minute extracted silk had an effect but a much smaller one - The 140°C oven resulted in a lower confidence interval of ~6000 Da.

Raw silk coils from silkworms were cut into pieces. The raw silk coil pieces were boiled in an aqueous solution of _Na₂CO₃ (approximately 100° _C ) for a period of approximately 30 to 60 minutes to remove the rubber (degumming). The volume of water used was approximately equal to 0.4 times the weight of the raw silk, and the _amount of _Na₂CO₃ was approximately 0.848 times the weight of the raw silk coil pieces. The resulting degummed silk coil pieces were rinsed three times with deionized water at approximately 60°C (20 minutes each). The volume of rinse water in each cycle was 0.2 L x the weight of the raw silk coil pieces. Excess water from the degummed silk coil pieces was removed. After the deionized water washing step, the wet degummed silk coil fragments are dried at room temperature. The degummed silk coil fragments are mixed with a LiBr solution and the mixture is heated to about 100°C. The heated mixture is placed in a drying oven and heated at a temperature in the range of about 60°C to about 140°C for about 60 minutes to achieve complete dissolution of the native silk protein. The resulting solution is cooled to room temperature and then dialyzed using a 3,500 Da MWCO membrane to remove the LiBr salt. Multiple exchanges are performed in deionized water until the ^Br- ion is less than 1 ppm, as measured in the hydrolyzed silk fiber protein solution read on an Oakton bromide ( ^Br- ) double junction ion selective electrode.

The resulting aqueous silk fibroin solution had a concentration of approximately 8.0% w/v and contained pure silk fibroin fragments having an average weight-average molecular weight selected from the group consisting of approximately 6 kDa to approximately 16 kDa, approximately 17 kDa to approximately 39 kDa, and approximately 39 kDa to approximately 80 kDa, and a polydispersity of approximately 1.5 to approximately 3.0. The 8.0% w/v solution was diluted with deionized water to provide coating solutions of 1.0% w/v, 2.0% w/v, 3.0% w/v, 4.0% w/v, and 5.0% w/v.

Various silk concentrations have been produced using tangential flow filtration (TFF). In all cases, a 1% silk solution was used as the input feed. Starting volumes of 1% silk solution ranged from 750-18,000 mL. The solution was filtered in TFF to remove lithium bromide. Once the residual LiBr level fell below a specified level, the solution was ultrafiltration to increase the concentration by removing water. See the example below.

Six (6) silk solutions were used in a standard silk configuration with the following results: Solution #1 had a silk concentration of 5.9 wt%, an average MW of 19.8 kDa, and a PDI of 2.2 (prepared using a 60-minute boiling extraction and a 1-hour LiBr dissolution at 100°C). Solution #2 had a silk concentration of 6.4 wt% (prepared using a 30-minute boiling extraction and a 4-hour LiBr dissolution at 60°C). Solution #3 had a silk concentration of 6.17 wt% (prepared using a 30-minute boiling extraction and a 1-hour LiBr dissolution at 100°C). Solution #4 had a silk concentration of 7.30 wt%: 7.30% silk solutions were generated starting with 30-minute extraction batches of 100 g silk coils. The extracted silk fibers were then dissolved in 9.3 M LiBr at 100°C in a 100°C oven for 1 hour. 100 g of silk fibers were dissolved in each batch to yield 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 15,500 mL of the 1% filtered silk solution was used as the starting volume/filter volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 1300 mL. 1262 mL of 7.30% silk was collected. Water was added to the feed to help remove excess solution, and 547 mL of 3.91% silk was collected. Solution #5 had a silk concentration of 6.44 wt%: 60-minute extraction batches of 25, 33, 50, 75, and 100 g of the silk mixture were started to produce a 6.44 wt% silk solution. The extracted silk fibers were then dissolved in 9.3 M LiBr at 100°C in a 100°C oven for 1 hour. Batches of 35, 42, 50, and 71 g of silk fibers were dissolved to produce 20% silk in LiBr and combined. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 17,000 mL of the 1% filtered silk solution was used as the starting volume/filter volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 3000 mL. 1490 mL of 6.44% silk was subsequently collected. Water was added to the feed to aid in the removal of residual solution, and 1454 mL of 4.88% silk was subsequently collected. Solution #6 had a silk concentration of 2.70 wt%: A 2.70% silk solution was produced starting with a 60-minute extraction batch of 25 g silk coils. The extracted silk fibers were then dissolved in a 100°C oven using 9.3 M LiBr at 100°C for 1 hour. 35.48 g of silk fibers were dissolved per batch to produce 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 1000 mL of the filtered 1% silk solution was used as the starting/filter volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 300 mL. 312 mL of 2.7% silk was then collected.

The preparation of fibroin solutions with higher molecular weight is given in Table O.

The silk waterborne coating compositions for application to fabrics are given in Tables P and Q below.

Three (3) silk solutions were used in membrane fabrication with the following results: Solution #1 had a silk concentration of 5.9%, an average MW of 19.8 kDa, and a PD of 2.2 (prepared by 60-minute boiling extraction and 1-hour dissolution in LiBr at 100°C). Solution #2 had a silk concentration of 6.4% (prepared by 30-minute boiling extraction and 4-hour dissolution in LiBr at 60°C). Solution #3 had a silk concentration of 6.17% (prepared by 30-minute boiling extraction and 1-hour dissolution in LiBr at 100°C).

Membranes were prepared according to Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published online September 22, 2011; doi:10.1038/nprot.2011.379). 4 mL of a 1% or 2% (wt/vol) silk solution in water was added to a 100 mm culture dish (the volume of silk can be varied to allow for thicker or thinner membranes and is not critical) and allowed to dry uncovered overnight. The bottom of a vacuum desiccator was filled with water. The dried membrane was placed in the desiccator and vacuum was applied, allowing the membrane to water anneal for 4 hours before removal from the culture dish. Membranes cast from Solution #1 did not produce a structurally continuous membrane; the membrane broke into several fragments. Despite water annealing, these membrane fragments remained soluble in water.

Solutions of various molecular weights and/or combinations of molecular weights can be optimized for gel applications. Examples of this process are provided below and are not intended to limit the applications or formulations. Three (3) silk solutions were used in gel preparation with the following results: Solution #1 had a silk concentration of 5.9%, an average MW of 19.8 kDa, and a PD of 2.2 (prepared using a 60 minute boiling extraction and a 1 hour LiBr solution at 100°C). Solution #2 had a silk concentration of 6.4% (prepared using a 30 minute boiling extraction and a 4 hour LiBr solution at 60°C). Solution #3 had a silk concentration of 6.17% (prepared using a 30 minute boiling extraction and a 1 hour LiBr solution at 100°C).

"Egel" is an electrocoagulation gelation process as described by Rockwood et al. Briefly, 10 ml of a silk-water solution was added to a 50 ml conical tube, and a pair of platinum wire electrodes were immersed in the silk solution. A 20 volt potential was applied to the platinum electrodes for 5 minutes, the power was turned off, and the gel was collected. Solution #1 did not form an EGEL within 5 minutes of applying the current.

Solutions #2 and #3 were gelled according to a published horseradish peroxidase (HRP) protocol. The behavior appeared typical of published solutions.

Materials and Methods: The following equipment and materials were used in the determination of silk molecular weight: Agilent 1100 equipped with ChemStation software version 10.01; refractive index detector (RID); analytical balance; volumetric flasks (1000 mL, 10 mL, and 5 mL); HPLC-grade water; ACS-grade sodium chloride; ACS-grade sodium hydrogen phosphate heptahydrate; phosphoric acid; dextran MW standards with nominal molecular weights of 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa; 50 mL PET or polypropylene disposable centrifuge tubes; graduated pipettes; amber glass HPLC vials with Teflon caps; and a Phenomenex PolySep GFC P-4000 column (dimensions: 7.8 mm × 300 mm). Procedure: A) Preparation of 1 L mobile phase (0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer)

Take a clean, dry 250 mL beaker, place it on a scale, and weigh it. Add approximately 3.3509 g of sodium biphosphate heptahydrate to the beaker. Record the exact weight of the sodium biphosphate weighed. Dissolve the weighed sodium phosphate by adding 100 mL of HPLC water to the beaker. Be careful not to spill any of the contents of the beaker. Carefully transfer the solution to a clean, dry 1000 mL volumetric flask. Rinse the beaker and transfer the rinse to the volumetric flask. Repeat the rinse 4-5 times. In a separate, clean, dry 250 mL beaker, accurately weigh approximately 5.8440 g of sodium chloride. Dissolve the weighed sodium chloride in 50 mL of water and transfer the solution to the sodium phosphate solution in the volumetric flask. Rinse the beaker and transfer the rinse solution to a volumetric flask. Adjust the pH of the solution to 7.0 ± 0.2 with phosphoric acid. Bring the volume of the flask to 1000 mL with HPLC water and shake vigorously to mix the solution. Filter the solution through a 0.45 μm polyamide membrane filter. Transfer the solution to a clean, dry solvent bottle and label the bottle. The volume of the solution can be adjusted as needed by adjusting the amounts of sodium hydrogen phosphate heptahydrate and sodium chloride accordingly. B) Preparation of Dextran Molecular Weight Standard Solution

Use at least five different molecular weight standards for each batch of samples so that the expected values of the test samples are within the values of the standards used. Label six 20 mL scintillation glass vials as molecular weight standards. Accurately weigh approximately 5 mg of each dextran molecular weight standard and record the weight. Dissolve the dextran molecular weight standard in 5 mL of mobile phase to prepare a 1 mg/mL standard solution. C) Sample Solution Preparation

When preparing sample solutions, if there are limitations on the amount of sample available, you can prepare proportionally as long as the ratios are maintained. Depending on the sample type and the silk protein content in the sample, weigh enough sample into a 50 mL disposable centrifuge tube on an analytical balance to prepare a 1 mg/mL sample solution for analysis. Dissolve the sample in an equal volume of mobile phase to make a 1 mg/mL solution. Cap the tube tightly and mix the sample (in solution). Incubate the sample solution at room temperature for 30 minutes. Gently mix the sample solution again for 1 minute and centrifuge at 4000 RPM for 10 minutes. D) HPLC Analysis of Samples

Transfer 1.0 mL of all standard and sample solutions to separate HPLC vials. Inject molecular weight standards (one injection each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions: E) Data Analysis and Calculation - Calculation of average molecular weight using Cirrus software

Upload the analytical data files of the standards and analytical samples to the Cirrus SEC data collection and molecular weight analysis software. Calculate the weight average molecular weight ( _Mw ), number average molecular weight ( _Mn ), peak average molecular weight ( _Mp ), and polydispersity of each injected sample.

Spider silk is a natural polymer composed of three domains: a repetitive central core domain that dominates the protein chain, and non-repetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, in which two basic sequences, crystalline (poly(A) or poly(GA)) and less crystalline (GGX or GPGXX) polypeptides, alternate. Drag silk is a protein complex composed of major stigmata protein 1 (MaSp1) and major stigmata protein 2 ( MaSp2 ). Both silks are approximately 3,500 amino acids long. MaSp1 is found in the fiber core and periphery, while MaSp2 forms clusters in certain core regions. The large central domains of MaSp1 and MaSp2 are organized in a block copolymer-like arrangement, with two basic sequences, crystalline (poly(A) or poly(GA)) and less crystalline (GGX or GPGXX) polypeptides, alternating within the core domain. Specific secondary structures have been assigned to the poly(A)/(GA), GGX, and GPGXX motifs, including β-sheets, α-helices, and β-helices, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain are responsible for the mechanical properties of spider silk; however, the non-repetitive N- and C-terminal domains are required for storage of the liquid silk stock in the lumen and for formation of the fiber system within the spinning conduit.

The primary difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which account for 15% of the total amino acid content in MaSp2 , while MaSp1 contains no proline. By counting the number of proline residues in the dragline silk of the clavipes spider ( N. clavipes ), it was possible to estimate the presence of two proteins in the fiber: 81% MaSp1 and 19% MaSp2 . Different spiders have different ratios of MaSp1 and MaSp2 . For example, dragline silk fibers from the golden garden spider contain 41% MaSp1 and 59% MaSp2 . These variations in the ratio of the main pot to the abdominal silk may determine the efficiency of the silk fiber.

One species of orb-weaving spider is known to have at least seven different types of silk proteins. Silks differ in their primary sequence, physical properties, and function. For example, drag silks, known for their use in constructing frameworks, radii, and lifelines, possess outstanding mechanical properties, including strength, toughness, and elasticity. Weight for weight, spider silk has higher toughness than steel and Kevlar. Flagellar silks, found in trapping spirals, have extensibility of up to 500%. Secondary potbelly silks, found in auxiliary spirals and prey wrappings of orb webs, have similarly high toughness and strength to primary potbelly silks, but do not supercontract in water.

Spider silk is renowned for its high tensile strength and toughness. Recombinant silk proteins also offer advantageous properties for cosmetic or dermatological compositions, particularly improved hydration or softening, good film-forming properties, and low surface density. Its diverse and unique biomechanical properties, combined with its biocompatibility and slow degradation rate, make spider silk an excellent candidate as a biomaterial for tissue engineering, guided tissue repair, and drug delivery, as well as for cosmetics (e.g., nail and hair enhancers, skincare products) and industrial materials (e.g., nanowires, nanofibers, surface coatings).

In one embodiment, the silk protein may include a polypeptide derived from a natural spider silk protein. The polypeptide is not particularly limited as long as it is derived from a natural spider silk protein, and examples of polypeptides include natural spider silk proteins and recombinant spider silk proteins, such as variants, analogs, derivatives or analogs thereof of natural spider silk proteins. In terms of excellent toughness, the polypeptide may be derived from the major drag silk protein produced in the spider's main pot gland. Examples of major drag silk proteins include the main pot gland spider silk proteins MaSp1 and MaSp2 from the rod-thread spider, and ADF3 and ADF4 from the cross garden spider ( Araneus diadematus ). Examples of polypeptides derived from major drag silk proteins include variants, analogs, derivatives or analogs thereof of major drag silk proteins. In addition, the polypeptide may be derived from the flagellar silk protein produced in the flagellar gland of the spider. Examples of flagellar silk proteins include flagellar silk proteins derived from the spider Nephila clavata and the like.

Examples of polypeptides derived from the major dragline protein include polypeptides containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, the polypeptide derived from the major dragline protein may be a polypeptide containing the amino acid sequence units represented by Formula 1: REP1-REP2 (1) and having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 at the C-terminus or an amino acid sequence having 90% or higher homology to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453. In the polypeptide derived from the major filament protein, the units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be identical to or different from each other. In the case of producing recombinant proteins using microorganisms such as Escherichia coli as a host, the molecular weight of the polypeptide derived from the major filament protein is 500 kDa or less, or 300 kDa or less, or 200 kDa or less in terms of productivity.

In formula (1), REP1 indicates polyalanine. In REP1, the number of alanine residues arranged continuously is preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and particularly preferably 5 or more. Furthermore, in REP1, the number of alanine residues arranged continuously is preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and particularly preferably 10 or less. In formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine, and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more of the total number of amino acid residues contained therein.

In the primary dragline, REP1 corresponds to the crystalline region of the fiber that forms the crystalline β-fold, while REP2 corresponds to the largely amorphous region of the fiber that lacks a regular structure and exhibits greater flexibility. Furthermore, [REP1-REP2] corresponds to the repetitive region (repeat sequence) composed of the crystalline and amorphous regions, which is a characteristic sequence of dragline proteins. Recombinant filament fragments

In some embodiments, the recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide, or a recombinant silk polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein include a recombinant spider silk polypeptide from the Araneidae or Araneoid families, or a recombinant insect silk polypeptide from the domestic silkworm. In some embodiments, the recombinant silk protein fragments disclosed herein include a recombinant spider silk polypeptide from the Theridiidae or Theridiidae families. In some embodiments, the recombinant silk protein fragments disclosed herein include a block copolymer comprising repeating units derived from a natural spider silk polypeptide from the Theridiidae or Theridiidae families. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having synthetic repeat units derived from spider silk polypeptides of the Theridiidae or Theridiidae families and non-repeat units derived from natural repeat units of spider silk polypeptides of the Theridiidae or Theridiidae families.

Recent advances in genetic engineering have provided pathways for producing a variety of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein produced heterologously in a prokaryotic or eukaryotic expression system using genetic engineering methods.

Various methods for synthesizing recombinant silk peptides are known and are described by Ausubel et al., Current Protocols in Molecular Biology § 8 (John Wiley & Sons 1987, (1990)), which is incorporated herein by reference. The Gram-negative bacterium Escherichia coli is a well-established host for industrial-scale protein production. Therefore, most recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively low cost, and can be scaled up for larger protein production.

Recombinant silk proteins can be produced by transforming prokaryotic or eukaryotic systems containing cDNA encoding silk proteins, fragments of these proteins, or analogs of these proteins. Recombinant DNA methods enable the production of recombinant silk with programmed sequence, secondary structure, structure, and precise molecular weight. There are four major steps in this process: (i) design and assembly of synthetic silk-like genes into gene "cassettes", (ii) insertion of these segments into recombinant DNA vectors, (iii) transformation of the recombinant DNA molecule into host cells, and (iv) expression and purification of selected clones.

As used herein, the term "recombinant vector" includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus or bacillary virus vectors) or artificial chromosome vectors, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors, and generally contain the desired coding sequence and the appropriate DNA sequences necessary for expressing the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast or plants) or in an in vivo expression system. Cloning vectors are generally used for engineering and amplification of certain desired DNA fragments and may lack the functional sequences required for expression of the desired DNA fragments.

Prokaryotic systems include Gram-negative or Gram-positive bacteria. Prokaryotic expression vectors may include an origin of replication recognized by the host organism, a homologous or heterologous promoter functional in the host, and a DNA sequence encoding a spider silk protein, a fragment of the protein, or a similar protein. Non-limiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum , Anabaena , Caulobacter , Gluconobacter , Rhodobacter , Pseudomonas , Paracoccus , Bacillus ( e.g. , Bacillus subtilis ) , Brevibacterium , Corynebacterium , Rhizobium . ( Meliloti ) , Flavobacterium , Klebsiella , Enterobacter , Lactobacillus , Lactococcus , Methylobacterium , Propionibacterium , Staphylococcus , or Streptomyces cells .

Eukaryotic systems include yeast and insect, mammalian, or plant cells. In this case, the expression vector may include a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding a spider silk protein, fragment, or analog, a polyadenylation sequence, a transcriptional termination site, and a final selection gene. Non-limiting examples of eukaryotic expression organisms include yeasts such as Saccharomyces cerevisiae , Pichia pastoris , basidiosporogenous yeast , and ascosporogenous yeast; filamentous fungi such as Aspergillus niger , Aspergillus oryzae , Aspergillus nidulans , Trichoderma reesei , Acremonium chrysogenum , Candida , Hansenula , and Kluyveromyces . , Saccharomyces ( e.g., brewer's yeast ) , Schizosaccharomyces , Pichia ( e.g., Pichia pastoris ) , or Yarrowia cells ; mammalian cells, such as HeLa cells, COS cells , and CHO cells; insect cells, such as Sf9 cells and MEL cells; and "insect host cells" such as Spodoptera frugiperda or Trichoplusia ni cells. SF9 cells, SF-21 cells, or High-Five cells , wherein SF-9 and SF-21 are ovary cells from the fall armyworm, and High-Five cells are egg cells from the cabbage looper; and "plant host cells," such as tobacco, potato, or pea cells.

A variety of heterologous host systems have been explored to produce various types of recombinant silk. Recombinant spider silk proteins and engineered silk have been cloned and expressed in bacteria (E. coli), yeast (Pichia pastoris), insects (silkworm larvae), plants (tobacco, soybean, potato, and Brassica juncea), mammalian cell lines (BHT/hamster), and transgenic animals (mice and goats). Most silk proteins carry an N- or C-terminal His-tag to simplify purification and yield sufficient protein.

In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include transgenic animals and plants. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include Escherichia coli. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include transgenic silkworms generated using genome editing technology (e.g., CRISPR).

The recombinant silk proteins disclosed herein comprise synthetic proteins based on repeat units of natural silk proteins. In addition to the synthetic repeat silk protein sequences, these sequences may further comprise one or more natural non-repeat silk protein sequences.

In some embodiments, "recombinant silk protein" refers to recombinant silk protein or fragments thereof. Recombinant production of silk protein and silk gel has been reported. A variety of hosts have been used for production, including E. coli, brewing yeast, Pseudomonas sp. , Rhodopseudomonas sp ., Bacillus sp. , and Strepomyces . See EP 0230702, which is incorporated herein by reference in its entirety.

Also provided herein are the design and biosynthesis of fibroin-like multiblock polymers comprising the GAGAGX hexapeptide (X is A, Y, V, or S) derived from the repeat domain of the heavy chain (H chain) of domestic silk.

In some embodiments, the present disclosure provides fibroin-like multiblock polymers derived from the repeat domain of the silkworm heavy chain (H chain), comprising GAGAGS hexapeptide repeating units. GAGAGS hexapeptide is the core unit of the H chain and plays an important role in the formation of crystallization domains. The fibroin-like multiblock polymers containing GAGAGS hexapeptide repeating units spontaneously aggregate into a β-pleated structure similar to native silk fibroin, wherein the fibroin-like multiblock polymers have any weight-average molecular weight described herein.

In some embodiments, the present disclosure provides a silk peptide-like multi-block copolymer composed of a GAGAGS hexapeptide repeating segment derived from the H chain of Bombyx motif silk heavy chain and a mammalian elastin VPGVG motif produced by Escherichia coli. In some embodiments, the present disclosure provides a fusion silk fibroin composed of a GAGAGS hexapeptide repeating segment derived from the H chain of Bombyx motif silk heavy chain and GVGVP produced by Escherichia coli, wherein the silk peptide-like multi-block copolymer has any weight average molecular weight described herein.

In some embodiments, the present disclosure provides a recombinant protein from Bombyx mori consisting of a (GAGAGS) _16- repeat segment. In some embodiments, the present disclosure provides a recombinant protein produced by Escherichia coli consisting of a (GAGAGS) _16- repeat segment and non-repeat (GAGAGS) ₁₆ -F-COOH, (GAGAGS) ₁₆ -FF-COOH, (GAGAGS) 16 -FFF-COOH, (GAGAGS) ₁₆ -FFFF-COOH, (GAGAGS) ₁₆ -FFFFFFFF-COOH, (GAGAGS) ₁₆ -FFFFFFFF-COOH, (GAGAGS) ₁₆ -FFFFFFFFFF-COOH, wherein F has the following amino acid sequence: SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG, and wherein the silk protein-like multiblock polymer has any weight average molecular weight described herein.

In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or a fragment thereof. The production of recombinant spider silk proteins based on partial cDNA clones has been reported. The recombinant spider silk protein thus produced comprises a portion of a repetitive sequence derived from the spider silk protein Spidroin 1 of the dragline silk of the spider Nephila spp. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87: 7120-7124 (1990). A cDNA clone encoding a portion of a repetitive sequence of Spidroin 2 , the second silk fiber protein of the dragline silk of the spider Nephila spp. and its recombinant synthesis are described in J. Biol. Chem. , 1992, Vol. 267, 19320-19324. Recombinant synthesis of spider silk proteins, including protein fragments and variants of the spider Nephila clavata, by transformant E. coli is described in U.S. Patent Nos. 5,728,810 and 5,989,894. cDNA clones encoding the spider silk protein from the schizont and their expression are described in U.S. Patent Nos. 5,733,771 and 5,756,6 77. A cDNA clone encoding a flagellar silk protein from an orb-web silk-spinning spider is described in U.S. Patent No. 5,994,099. U.S. Patent No. 6,268,169 describes the recombinant synthesis of a spider silk-like protein derived from a repetitive peptide sequence found in native spider silk from the spider Nephila clavata using an Escherichia coli, Bacillus subtilis, and Pichia pastoris recombinant expression system. WO 03/020916 describes cDNA clones encoding and recombinantly producing spider silk proteins having cDNAs derived from the main potbelly glands of Nephila madagascariensis , Nephila senegalensis , Tetragnatha kauaiensis , Tetragnatha versicolor , Argiope aurantia , Argiope trifasciata, Gasteracantha mammosa , and Latrodectus geometricus , the flagellar glands of Argiope trifasciata , the potbelly glands of Dolomedes tenebrosus , the flagellar glands of Plectreurys striata, and the flagellar glands of Nephila madagascariensis. tristis and the silk glands of the mygalomorph Euagrus chisoseus . Each of the above references is incorporated herein by reference in its entirety.

In some embodiments, the recombinant spider silk protein is a hybrid of spider silk protein and insect silk protein, a hybrid of spider silk protein and collagen, a hybrid of spider silk protein and arthropod elastic protein, or a hybrid of spider silk protein and keratin. The spider silk repeat unit comprises or consists of an amino acid sequence of a region comprising or consisting of at least one peptide motif that is repeated in naturally occurring major pot-shaped gland polypeptides, such as dragging silk polypeptides, secondary pot-shaped gland polypeptides, flagellin polypeptides, aggregating silk polypeptides, grape-shaped silk polypeptides, or piriform silk polypeptides.

In some embodiments, the recombinant spider silk proteins disclosed herein comprise synthetic spider silk proteins derived from repeat units, consensus sequences, and, optionally, one or more naturally occurring non-repetitive spider silk protein sequences of a natural spider silk protein. The repeat units of a natural spider silk polypeptide may include a dragline silk polypeptide or a flagellate spider silk polypeptide of the family Theridiidae or Theridiidae.

As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that is repeated in a naturally occurring major diatom polypeptide, such as a dragline, secondary diatom polypeptide, flagellate, aggregating, grape-like, or piriform silk polypeptide. A "repeat unit" refers to a region in an amino acid sequence that corresponds to a region comprising or consisting of at least one peptide motif (e.g., AAAAAA or GPGQQ) that is repeated in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., a consistent amino acid sequence) or a substantially similar amino acid sequence (i.e., a variant amino acid sequence). A "repeat unit" having an amino acid sequence that is "substantially similar" to the corresponding amino acid sequence in a naturally occurring silk polypeptide (i.e., a wild-type repeat unit) is also similar in its properties. For example, a silk protein comprising a "substantially similar repeat unit" remains insoluble and maintains its insolubility. For example, a "repeat unit" having an amino acid sequence that is "identical" to the amino acid sequence of a naturally occurring silk polypeptide can be a portion of a silk polypeptide that corresponds to one or more peptide motifs of MaSpI, MaSpII, ADF-3, and/or ADF-4. For example, a "repeat unit" having an amino acid sequence that is "substantially similar" to the amino acid sequence of a naturally occurring silk polypeptide can be a portion of a silk polypeptide that corresponds to one or more peptide motifs of MaSpI, MaSpII, ADF-3, and/or ADF-4 but has one or more amino acid substitutions at specific amino acid positions.

As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing a frequently occurring amino acid (e.g., "G") at a certain position, in which other unidentified amino acids are replaced by the placeholder "X." In some embodiments, the consensus sequence is at least one of the following: (i) GPGXX, wherein X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, wherein X is an amino acid selected from Y, P, R, S, A, T, N, and Q, preferably Y, P, and Q; or (iii) A _x , wherein x is an integer from 5 to 10.

The consensus peptide sequences GPGXX and GGX, i.e., glycine-rich motifs, provide flexibility to silk polypeptides and, therefore, to silk threads formed from silk proteins containing these motifs. In detail, repeated GPGXX motifs form a turn-helical structure that confers elasticity to silk polypeptides. Both the main pot abdominal silk and the flagellar silk have GPGXX motifs. Repeated GGX motifs are associated with a helical structure with three amino acids per turn and are found in most spider silks. The GGX motif can provide additional elastic properties to silk. Repeated polyalanine Ax (peptide) motifs form a crystalline β-pleated structure that provides strength to silk polypeptides, as described, for example, in WO 03/057727.

In some embodiments, the recombinant spider silk proteins disclosed herein comprise two identical repeat units, each of which comprises at least one, preferably one, amino acid sequence selected from the group consisting of GGRPSDTYG and GGRPSSSYG, derived from elastin. elastin is an elastic protein found in most arthropods that provides low stiffness and high strength.

As used herein, a "non-repetitive unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repetitive (carboxyl-terminal) amino acid sequence in a naturally occurring dragline silk polypeptide (i.e., a wild-type non-repetitive (carboxyl-terminal) unit), preferably ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2), NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO: 42), and the C16 peptide comprising 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP described in U.S. Patent No. 8,367,803 ( spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk), an amino acid sequence adapted from the native sequence of ADF4 from the spider Araneus diadematus. The non-repetitive ADF-4 and its variants showed efficient assembly behavior.

Among synthetic spider silk proteins, the recombinant silk proteins disclosed herein, in some embodiments, include a C16 protein having the polypeptide sequence SEQ ID NO: 1 as described in U.S. Patent No. 8288512. In addition to the polypeptide sequence shown in SEQ ID NO: 1, functional equivalents, functional derivatives, and salts of this sequence are also specifically included.

As used herein, "functional equivalents" refer to mutants having an amino acid other than the specifically mentioned amino acid in at least one sequence position of the above amino acid sequence.

In some embodiments, the recombinant spider silk protein disclosed herein comprises an effective amount of at least one natural or recombinant silk protein, including a Spidroin major 1 described by Xu et al., PNAS, USA, 87, 7120, (1990), a Spidroin major 2 described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), or a Spidroin major 3 described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922). 2, such as the recombinant spider silk proteins described in U.S. Patent Application No. 2016/0222174 and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,803, 8,097,583, 8,030,024, 7,754,851, 7,148,039, and 7,060,260, or alternatively, the minor spider silk proteins described in patent application WO 95/25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spider silk proteins suitable for use in the recombinant RSPF disclosed herein include ADF3 and ADF4 from the aponeurotic gland of the spider Araneus cruciferus.

Reorganized yarns are also described in other patents and patent applications incorporated herein by reference: US 2004590196, US 7,754,851, US 2007654470, US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US 200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542、US 2009265344、US 2012684607、 US 2004583227, US 8,030,024, US 2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US 20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US 2012697729, US 20150328363, US 9,034,816, US 20130172478, US 9,217,017, US 20170202995, US 8,721,991, US 2008227498, US 9,233,067, US 8,288,512, US 2008161364, US 7,148,039, US 1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902、US 7,569,660、 US 1999138833, US 2000591632, US 20120065126, US 20100278882, US 2008161352, US 20100015070, US 2009513709, US 20090194317, US 2004559286, US 200589551, US 2008187824, US 20050266242, US 20050227322 and US 20044418.

Reorganized yarns are also described in other patents and patent applications incorporated herein by reference: US 20190062557, US 20150284565, US 20130225476, US 20130172478, US 20130136779, US 20130109762, US 20120252294, US 20110230911, US 20110201783, US 20100298877, US 10,478,520, US 10,253,213, US 10,072,152, US 9,233,067, US 9,217,017, US 9,034,816, US 8,877,903, US 8,729,238, US 8,721,991, US 8,097,583, US 8,034,897, US 8,030,024, US 7,951,908, US 7,868,146 and US 7,754,851.

In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of 2 to 80 repeat units, each of which is independently selected from GPGXX, GGX, and _Ax as defined herein.

In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of repeat units, each of which is independently selected from the group consisting of GPGAS, GPGSG, GPGGY, GPGGP, GPGGA, GPGQQ, GPGGG, GPGQG, GPGGS, GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA, AAAAAA, AAAAAAA, AAAAAAAAA, AAAAAAAAAA, GGRPSDTYG, and GGRPSSSYG as described in U.S. Patent No. 8,877,903, (i) GPYGPGASAAAAAAGGYGPGSGQQ, (ii) GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP, (iii) GPGQQGPGQQGPGQQGPGQQ, (iv) GPGGAGGPYGPGGAGGPYGPGGAGGPY, (v) GGTTIIEDLDITIDGADGPITISEELTI, (vi) PGSSAAAAAAAAASGPGQGQGQGQGQGGRPSDTYG, (vii) SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG, (viii) GGAGGAGGAGGSGGAGGS, (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY, (x) GPYGPGASAAAAAAGGYGPGCGQQ, (xi) GPYGPGASAAAAAAGGYGPGKGQQ, (xii) GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP ,(xiii) GSSAAAAAAAASGPGGYGPKNQGSGPGGYGPGGP, (xiv) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP or variants thereof, such as synthetic spider peptides having GPGAS, GGY, GPGSG in the order of the peptide chain, or AAAAAAAA, GPGGY, GPGGP in the order of the peptide chain, or AAAAAAAA, GPGQG, GGR in the order of the peptide chain.

In some embodiments, the present disclosure provides spidroin-like multi-block peptides that mimic amino acid repeat units derived from natural spider silk proteins (such as spidroin major 1 domain, spidroin major 2 domain, or spidroin minor 1 domain) and the variation profile between repeat units without changing their three-dimensional structure, wherein these spidroin-like multi-block peptides comprise amino acid repeat units corresponding to one of the following sequences (I), (II), (III), and/or (IV). [(XGG) _w (XGA)(GXG) _x (AGA) _y (G) _z AG] _p Formula (I), wherein: X corresponds to tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer and has any weight average molecular weight described herein, and/or [(GPG ₂ YGPGQ ₂ ) _a (X') ₂ S(A) _b ] _p Formula (II), wherein: X' corresponds to the amino acid sequence GPS or GPG, a is 2 or 3, b is an integer from 7 to 10, and p is an integer and has any weight average molecular weight described herein, and/or [(GR)(GA) _l (A) _m (GGX) _n (GA) _l (A) _m ] _p Formula (III) and/or [(GGX) _n (GA) _m (A) _l ] _pFormula (IV), wherein: X'' corresponds to tyrosine, glutamine or alanine, l is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer.

In some embodiments, the recombinant spider silk protein or an analog of a spider silk protein comprises amino acid repeat units of sequence (V): [(Xaa Gly Gly) _w (Xaa Gly Ala)(Gly Xaa Gly) _x (Ala Gly Ala) _y (Gly) _z Ala Gly] _p formula (V), wherein Xaa is tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer.

In some embodiments, the recombinant spider silk protein of the present disclosure is selected from the group consisting of ADF-3 or a variant thereof, ADF-4 or a variant thereof, MaSpI (SEQ ID NO: 43) or a variant thereof, MaSpII (SEQ ID NO: 44) or a variant thereof, as described in U.S. Patent No. 8,367,803.

In some embodiments, the present disclosure provides water-soluble recombinant spider silk proteins produced in mammalian cells. The solubility of spider silk proteins produced in mammalian cells is attributed to the presence of a COOH-terminal amino acid in these proteins, which renders them more hydrophilic. These COOH-terminal amino acids are absent in spider silk proteins expressed in microbial hosts.

In some embodiments, the recombinant spider silk protein disclosed herein comprises a water-soluble recombinant spider silk protein C16 modified with an amino or carboxyl terminus selected from an amino acid sequence consisting of: GCGGGGGG, GKGGGGGG, GCGGSGGGGSGGGG, GKGGGGGGSGGGG, and GCGGGGGGSGGGG. In some embodiments, the recombinant spider silk protein disclosed herein comprises _C16NR4 , _C32NR4 , C16, _C32 , _NR4C16NR4 , NR4C32NR4, _NR3C16NR3 , or _NR3C32NR3 , such that the molecular weight of the protein is within the range described herein.

In some embodiments, the recombinant spider silk protein disclosed herein comprises a recombinant spider silk protein having a synthetic repeat peptide segment and an amino acid sequence adapted from the native sequence of ADF4 from the spider Araneus striata, as described in U.S. Patent No. 8,877,903. In some embodiments, the RSPF disclosed herein comprises a recombinant spider silk protein having a repeat peptide unit derived from a native spider silk protein (such as the spidroin major 1 domain, spidroin major 2 domain, or spidroin minor 1 domain), wherein the repeat peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG, as described in U.S. Patent No. 8,367,803.

In some embodiments, the present disclosure provides recombinant spider proteins composed of GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY repeating segments and having a molecular weight as described herein.

As used herein, the term "recombinant silk" refers to recombinant spider and/or silk proteins or fragments thereof. In one embodiment, the spider silk protein is selected from the group consisting of: sheath silk (grape silk), egg capsule silk (cylindrical silk), egg shell silk (tubular silk), non-adhesive drag silk (potential silk), attachment thread silk (pyriform silk), adhesive core fiber (flagellate silk), and adhesive outer fiber (aggregate silk). For example, recombinant spider silk proteins described herein include proteins described in U.S. Patent Application No. 2016/0222174 and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734.

Some organisms produce a variety of silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb-weaving spiders have six distinct types of glands that produce different silk polypeptide sequences that polymerize into fibers tailored to their environment or niche in the life cycle. Fibers are named after the gland from which they originate, and polypeptides are labeled with the gland's abbreviation (e.g., "Ma") and "Sp," short for spider silk protein. In orb-weaving spiders, these types include major potbelly (MaSp, also called dragline), minor potbelly (MiSp), flagellar (Flag), grape-shaped (AcSp), tubular (TuSp), and piriform (PySp). This combination of polypeptide sequence variation across fiber types, domains, and between different genera and species of organisms results in a wealth of potential properties that can be exploited through the commercial production of recombinant fibers. To date, the vast majority of research on recombinant silk has focused on the major bowel spidroin (MaSp) protein.

Acroporous (AcSp) filaments tend to have high toughness, resulting from a combination of moderately high strength and moderately high elongation. AcSp filaments are characterized by large block ("bulk repeat") size and are typically incorporating polyserine and GPX motifs. Tubular (TuSp or cylindrical) filaments tend to have large diameters, with moderate strength and high elongation. TuSp filaments are characterized by their polyserine and polythreonine content, as well as short segments of polyalanine. Main belly (MaSp) filaments tend to have high strength and moderate elongation. MaSp filaments can be one of two subtypes: MaSp1 and MaSp2. MaSp1 filaments are generally less elongated than MaSp2 filaments and are characterized by polyalanine, GX, and GGX motifs. MaSp2 filaments are characterized by polyalanine, GGX, and GPX motifs. MiSp filaments tend to be of medium strength and medium ductility. MiSp filaments are characterized by GGX, GA, and polyA motifs and typically contain spacer elements of approximately 100 amino acids. Flagellar (Flag) filaments tend to be very ductile and of medium strength. Flag filaments are typically characterized by GPG, GGX, and short spacer motifs.

Silk polypeptides are characteristically composed of a repeat domain (REP) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids long. The repeat domain exhibits a hierarchical structure. The repeat domain comprises a series of blocks (also known as repeat units). These blocks are repeated throughout the silk repeat domain, sometimes completely and sometimes incompletely (forming quasi-repeated domains). The length and composition of the blocks vary between different silk types and between different species. Table 1 of U.S. Published Application No. 2016/0222174 (incorporated herein in its entirety) lists examples of block sequences from selected species and silk types. Additional examples are presented in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pp. 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, blocks can be arranged in a regular pattern, forming larger, mega-repeated sequences that occur multiple times (typically 2-8 times) within the repeat domain of the silk sequence. Repeat blocks within a repeat domain or large repeat sequence and repeat large repeat sequences within a repeat domain can be separated by spacer elements.

According to certain embodiments of the present disclosure, certain spider silk block copolymer polypeptides constructed from block and/or large repeat domains are described in U.S. Published Patent Application No. 2016/0222174.

Recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant polypeptide is expressed and subsequently secreted from the host cell for easy purification from the surrounding culture medium. If an expression/secretion vector is not used, an alternative method involves purifying the recombinant block copolymer polypeptide from a cell lysate (the cell residue after disruption of cell integrity) derived from the prokaryotic or eukaryotic cells expressing the polypeptide. Methods for producing such cell lysates are known to those skilled in the art. In some embodiments, the recombinant block copolymer polypeptide is isolated from the cell culture supernatant.

Recombinant block copolymer polypeptides can be purified by affinity separation, such as by immunological interaction with antibodies that specifically bind to the recombinant polypeptide or by using a nickel column to separate recombinant polypeptides labeled with 6-8 histidine residues at the N- or C-terminus. Alternative tags can include the FLAG epitope or the hemagglutinin epitope. Such methods are typically used by those skilled in the art.

Solutions of such polypeptides (ie, recombinant silk proteins) can then be prepared and used as described herein.

In another embodiment, recombinant silk protein can be prepared according to the method described in U.S. Patent No. 8,642,734 (the entire contents of which are incorporated herein) and used as described herein.

In one embodiment, a recombinant spider silk protein is provided. Spider silk proteins typically consist of 170 to 760 amino acid residues, such as 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, such as 300 to 400 amino acid residues, and more preferably 340 to 380 amino acid residues. A small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require the use of harsh solvents for dissolution and polymerization. The recombinant spider silk protein may contain more than 760 residues. In particular, when the spider silk protein contains more than two fragments derived from the N-terminal portion of the spider silk protein, the spider silk protein comprises an N-terminal fragment composed of at least one fragment (NT) derived from the corresponding portion of the spider silk protein and a repeat fragment (REP) derived from the corresponding internal fragment of the spider silk protein. Optionally, the spider silk protein comprises a C-terminal fragment (CT) derived from the corresponding fragment of the spider silk protein. The spider silk protein typically comprises a single fragment (NT) derived from the N-terminal portion of the spider silk protein, but in a preferred embodiment, the N-terminal fragment comprises at least two, for example two, fragments (NT) derived from the N-terminal portion of the spider silk protein. Thus, spider silk proteins can be schematically represented by the formula NT _m -REP, and alternatively NT _m -REP-CT, where m is an integer of 1 or greater, such as 2 or greater, and preferably is in the range of 1-2, 1-4, 1-6, 2-4, or 2-6. Preferred spider silk proteins can be schematically represented by the formula NT ₂ -REP or NT-REP, and alternatively NT ₂ -REP-CT or NT-REP-CT. Protein fragments are typically covalently coupled via peptide bonds. In one embodiment, the spider silk protein consists of an NT segment coupled to a REP segment, which is optionally coupled to a CT segment.

In one embodiment, the first step in a method for producing isolated spider silk protein polymers involves expressing a polynucleic acid molecule encoding the spider silk protein in a suitable host, such as Escherichia coli. The resulting protein is isolated using standard procedures. Optionally, lipopolysaccharides and other pyrogens are actively removed during this stage.

In the second step of the method for producing polymers of isolated spider silk proteins, a solution of the spider silk protein in a liquid medium is provided. The terms "soluble" and "in solution" mean that the protein does not visibly aggregate at 60,000 × g and does not precipitate from the solvent. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as 10-50 mM Tris-HCl buffer or a phosphate buffer. The liquid medium has a pH of 6.4 or higher and/or contains an ionic composition that prevents the polymerization of the spider silk protein. That is, the liquid medium has a pH of 6.4 or higher, or contains an ionic composition that prevents the polymerization of the spider silk protein, or both.

Ionic compositions that prevent spider silk protein polymerization can be readily prepared by skilled artisans using the methods disclosed herein. Preferred ionic compositions that prevent spider silk protein polymerization have an ionic strength greater than 300 mM. Specific examples of ionic compositions that prevent spider silk protein polymerization include greater than 300 mM NaCl, 100 mM phosphate, and combinations of these ions that have the desired preventive effect on spider silk protein polymerization, such as a combination of 10 mM phosphate and 300 mM NaCl.

The presence of the NT fragment improves the stability of the solution and prevents the formation of polymers under these conditions. This can be advantageous when immediate polymerization may be undesirable, such as during protein purification, in large-scale preparations, or when other conditions need to be optimized. The pH of the liquid medium is preferably adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as up to 10.5, to achieve high solubility of the spider silk protein. It can also be advantageous to adjust the pH of the liquid medium to a range of 6.4-6.8, which provides sufficient solubility of the spider silk protein, but facilitates subsequent adjustment of the pH to 6.3 or lower.

In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or lower and an ionic composition that allows polymerization. That is, if the liquid medium in which the spider silk protein is dissolved has a pH of 6.4 or higher, the pH is lowered to 6.3 or lower. The skilled person is familiar with various ways to achieve this, which generally involve adding strong or weak acids. If the liquid medium in which the spider silk protein is dissolved has an ionic composition that prevents polymerization, the ionic composition is changed to allow polymerization. The skilled person is familiar with various ways to achieve this, such as dilution, dialysis or gel filtration. If necessary, this step involves lowering the pH of the liquid medium to 6.3 or lower and changing the ionic composition to allow polymerization. Preferably, the pH of the liquid medium is adjusted to 6.2 or lower, such as 6.0 or lower. In particular, from a practical perspective, it may be advantageous to lower the pH from 6.4 or 6.4-6.8 in the previous step to 6.3 or 6.0-6.3 (e.g., 6.2) in this step. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range, e.g., 4.2-6.3, promotes rapid polymerization.

In the fourth step, the spider silk protein is polymerized in a liquid medium having a pH of 6.3 or lower and an ionic composition that permits spider silk protein polymerization. Although the presence of the NT fragment improves the solubility of the spider silk protein at a pH of 6.4 or higher and/or in an ionic composition that prevents spider silk protein polymerization, the fragment accelerates polymer formation at a pH of 6.3 or lower when the ionic composition permits spider silk protein polymerization. The resulting polymer is preferably solid and macroscopic, and is formed in a liquid medium having a pH of 6.3 or lower and an ionic composition that permits spider silk protein polymerization. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range (e.g., 4.2-6.3) promotes rapid polymerization, and the resulting polymers can be provided at the molecular weights described herein and prepared in solution form, which can be used for coating of articles as desired.

Ionic compositions that allow spider silk protein polymerization can be readily prepared by a skilled artisan using the methods disclosed herein. Preferred ionic compositions that allow spider silk protein polymerization have an ionic strength of less than 300 mM. Specific examples of ionic compositions that allow spider silk protein polymerization include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack a preventive effect on spider silk protein polymerization, such as a combination of 10 mM phosphate or 20 mM phosphate with 150 mM NaCl. The ionic strength of the liquid medium is preferably adjusted to a range of 1-250 mM.

Without wishing to be bound by any particular theory, it is hypothesized that the NT segments have oppositely charged polarities and that environmental changes in pH affect the charge balance on the protein surface, followed by aggregation, while salt inhibits the same event.

At neutral pH, the energetic cost of burying the excess negative charge of the acidic poles would be expected to prevent aggregation. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces eventually become dominant, explaining the observed salt- and pH-dependent polymerization behavior of NTs and NT-containing micro-spider silk proteins. It is proposed that, in some embodiments, pH-induced NT polymerization and increased fiber assembly efficiency of NT-micro-spider silk proteins are attributed to changes in surface electrostatic potential, with the accumulation of acidic residues at one of the NT poles shifting its charge balance, causing the polymerization transition to occur at pH values of 6.3 or lower.

In the fifth step, the resulting preferably solid spider silk protein polymer is separated from the liquid medium. Optionally, this step involves the active removal of lipopolysaccharides and other pyrogens from the spider silk protein polymer.

Without wishing to be bound by any particular theory, it has been observed that the formation of spider silk protein polymers proceeds through the formation of water-soluble spider silk protein dimers. Therefore, the present disclosure also provides a method for producing isolated spider silk protein dimers, wherein the first two method steps are as described above. The spider silk protein exists as a dimer in a liquid medium with a pH of 6.4 or higher and/or an ionic composition that prevents the polymerization of the spider silk protein. The third step involves isolating the dimer obtained in the second step and, if appropriate, removing lipopolysaccharides and other pyrogens. In a preferred embodiment, the spider silk protein polymers of the present disclosure are composed of polymerized protein dimers. Therefore, the present disclosure provides novel uses of spider silk proteins, preferably those spider silk proteins disclosed herein, for producing spider silk protein dimers.

According to another aspect, the present disclosure provides polymers of spider silk proteins as disclosed herein. In one embodiment, polymers of this protein can be obtained by any of the methods disclosed herein for use therein. Therefore, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing polymers of spider silk proteins as coatings based on recombinant silk. According to one embodiment, the present disclosure provides novel uses of dimers of spider silk proteins, preferably those disclosed herein, for producing polymers of isolated spider silk proteins as coatings based on recombinant silk. In these uses, preferably, the polymer is produced in a liquid medium having a pH of 6.3 or lower and an ionic composition that allows the polymerization of the spider silk protein. In one embodiment, the pH of the liquid medium is 3 or higher, such as 4.2 or higher. The resulting pH range, such as 4.2-6.3, promotes rapid polymerization.

Using the methods disclosed herein, the polymerization process can be controlled, and this allows optimization of parameters for obtaining silk polymers with desired properties and shapes.

In one embodiment, the recombinant silk proteins described herein include those described in U.S. Patent No. 8,642,734, the entire contents of which are incorporated by reference.

In another embodiment, the recombinant silk proteins described herein can be prepared according to the methods described in U.S. Patent No. 9,051,453, the entire contents of which are incorporated herein by reference.

The amino acid sequence represented by SEQ ID NO: 1 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence consisting of 50 amino acid residues from the C-terminal amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI: 1263287). The amino acid sequence represented by SEQ ID NO: 2 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 of U.S. Patent No. 9,051,453 with 20 residues removed from the C-terminus. The amino acid sequence represented by SEQ ID NO: 3 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 with 29 residues removed from the C-terminus.

An example of a polypeptide containing an amino acid sequence unit represented by Formula 1: REP1-REP2(1) and having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 at the C-terminus, or an amino acid sequence having 90% or higher homology with an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 is a polypeptide having an amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453. The polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453 was obtained by the following mutations: An amino acid sequence consisting of a start codon, a His 10 tag, and a HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) was added to the N-terminus of the ADF3 amino acid sequence (NCBI Accession No. AAC47010, GI: 1263287). The first to 13 repeat regions were approximately doubled, and the translation terminated at amino acid residue 1154. In the polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 3.

In addition, a polypeptide containing an amino acid sequence unit represented by Formula 1: REP1-REP2 (1) and having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 at the C-terminus, or an amino acid sequence having 90% or higher homology to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 may be a protein having an amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, wherein one or more amino acids have been substituted, deleted, inserted and/or added and having a repeating region consisting of a crystalline region and an amorphous region.

In addition, an example of a polypeptide containing two or more amino acid sequence units represented by Formula 1: REP1-REP2 (1) is a recombinant protein derived from ADF4, which has an amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051,453 is an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of a partial amino acid sequence of ADF4 obtained from the NCBI database (NCBI Accession No.: AAC47011, GI: 1263289). In addition, a polypeptide containing two or more amino acid sequence units represented by Formula 1: REP1-REP2 (1) may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and/or added and has a repeat region consisting of a crystalline region and an amorphous region. In addition, an example of a polypeptide containing two or more amino acid sequence units represented by Formula 1: REP1-REP2 (1) is a recombinant protein derived from MaSp2, which has an amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453 is obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of a partial sequence of MaSp2 obtained from the NCBI online database (NCBI Accession No.: AAT75313, GI: 50363147). In addition, a polypeptide containing two or more amino acid sequence units represented by Formula 1: REP1-REP2 (1) may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453, wherein one or more amino acids have been substituted, deleted, inserted and/or added and has a repeating region consisting of a crystalline region and an amorphous region.

Examples of polypeptides derived from flagellin include polypeptides containing 10 or more amino acid sequence units represented by Formula 2: REP3(2), preferably polypeptides containing 20 or more units thereof, and more preferably polypeptides containing 30 or more units thereof. In the case of producing recombinant proteins using microorganisms such as Escherichia coli as hosts, the molecular weight of the polypeptide derived from flagellin is preferably 500 kDa or less, more preferably 300 kDa or less, and further preferably 200 kDa or less in terms of productivity.

In formula (2), REP 3 represents an amino acid sequence consisting of Gly-Pro-Gly-Gly-X, wherein X represents an amino acid selected from the group consisting of Ala, Ser, Tyr, and Val.

A key characteristic of spider silk is that flagellar filaments lack crystalline regions and instead have repetitive regions composed of amorphous regions. Because primary filaments and their analogs have repetitive regions composed of crystalline and amorphous regions, they are expected to exhibit high stress and stretchability. Meanwhile, flagellar filaments exhibit high stretchability despite lower stress than primary filaments. This is believed to be because the majority of flagellar filaments are composed of amorphous regions.

An example of a polypeptide containing 10 or more amino acid sequence units represented by Formula 2: REP3(2) is a recombinant protein derived from flagellar filament protein having an amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9,051,453. The amino acid sequence represented by 19 was obtained by combining the partial sequence of the flagellar silk protein of the spider Nephila clavata obtained from the NCBI database (NCBI accession number: AAF36090, GI: 7106224), specifically the amino acid sequence corresponding to the repeat segment and motif from the 1220th residue to the 1659th residue from the N-terminus (referred to as the PR1 sequence), and the partial sequence of the flagellar silk protein of the spider Nephila clavata obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649), specifically the C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus, and then replacing the start codon, His An amino acid sequence obtained by adding an amino acid sequence consisting of a 10-tag and an HRV3C protease recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of the combined sequence. In addition, a polypeptide containing 10 or more amino acid sequence units represented by Formula 2: REP3 (2) may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9,051,453, wherein one or more amino acids have been substituted, deleted, inserted and/or added and has a repeat region consisting of an amorphous region.

The polypeptide can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, and the gene can be produced by amplifying a gene encoding a natural spider silk protein from spider-derived cells using polymerase chain reaction (PCR) or the like and cloning it, or by chemical synthesis. Furthermore, the method for chemically synthesizing the gene is not particularly limited, and the gene can be synthesized, for example, by ligating oligonucleotides automatically synthesized using AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) using PCR or the like based on information on the amino acid sequence of a natural spider silk protein obtained from the NCBI online database or the like. At this time, in order to facilitate protein purification and observation, a gene encoding a protein having the above-mentioned amino acid sequence can be synthesized, with an amino acid sequence consisting of a start codon and a His 10 tag added to the N-terminus of the amino acid sequence.

Examples of expression vectors include plasmids, bacteriophages, viruses, and the like that can express proteins based on DNA sequences. Plasmid-type expression vectors are not particularly limited, as long as they allow the target gene to be expressed in host cells and can be amplified. For example, when using Escherichia coli Rosetta (DE3) as a host, pET22b(+) plasmid vectors, pCold plasmid vectors, and the like can be used. Of these, pET22b(+) plasmid vectors are preferred in terms of protein productivity. Examples of hosts include animal cells, plant cells, microorganisms, and the like.

The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, one of the two major silk proteins of the spider A. cross. This polypeptide has the advantages of having high strength-elongation and toughness and being easy to synthesize.

Thus, recombinant silk proteins (e.g., proteins based on recombinant spider silk) used in accordance with the embodiments, articles, and/or methods described herein may include those described above or in U.S. Patent Nos. 8,173,772, 8,278,416, 8,618,255, 8,642,734, 8,691,581, 8,729,235, 9,115,204, 9,157,070, 9,309,299, 9,644,012, 9,708,376, 9,051,45 3, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332; and U.S. Patent Publication Nos. 2009/0226969, 2011/0281273, 2012/0041177, 2013/0065278, 2013/0115698, and No. 2013/0316376, No. 2014/0058066, No. 2014/0079674, No. 2014/0245923, No. 2015/0087046, No. No. 2015/0119554, No. 2015/0141618, No. 2015/0291673, No. 2015/0291674, No. 2015/0239587, No. No. 2015/0344542, No. 2015/0361144, No. 2015/0374833, No. 2015/0376247, No. 2016/0024464, No. No. 2017/0066804, No. 2017/0066805, No. 2015/0293076, No. 2016/0222174, No. 2017/0283474, No. No. 2017/0088675, No. 2019/0135880, No. 2015/0329587, No. 2019/0040109, No. 2019/0135881, No. No. 2019/0177363, No. 2019/0225646, No. 2019/0233481, No. 2019/0031842, No. 2018/0355120, No. No. 2019/0186050, No. 2019/0002644, No. 2020/0031887, No. 2018/0273590, No. 20191/094403, No. 2019/0031843, No. 2018/0251501, No. 2017/0066805, No. 2018/0127553, No. 2019/0329526, No. 2020/0031886, No. 2018/0080147, No. 2019/0352349, No. 2020/0043085, No. 2019/0144819, No. One or more recombinant silk proteins described in Patents Nos. 2019/0228449, 2019/0340666, 2020/0000091, 2019/0194710, 2019/0151505, 2018/0265555, 2019/0352330, 2019/0248847, and 2019/0378191, the entire contents of which are incorporated herein by reference. Fibroin-like protein fragments

The recombinant silk proteins disclosed herein comprise synthetic proteins based on repeat units of native silk proteins. In addition to synthetic repeat silk protein sequences, these sequences may further comprise one or more native non-repeat silk protein sequences. As used herein, "fibroin-like protein fragments" refer to protein fragments having a molecular weight and polydispersity as defined herein and having a degree of homology to a protein selected from native silk proteins, fibroin heavy chains, fibroin light chains, or any protein comprising one or more GAGAGS hexaamino acid repeat units. In some embodiments, the degree of homology is selected from about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, or less than 75%.

As described herein, proteins such as protofilaments, filoprotein heavy chain, filoprotein light chain, or any protein comprising one or more GAGAGS hexaamino acid repeat units include about 9% to about 45% glycine, or about 9% glycine, or about 10% glycine, about 43% glycine, about 44% glycine, about 45% glycine, or about 46% glycine. As described herein, proteins such as protofilaments, filoprotein heavy chain, filoprotein light chain, or any protein comprising one or more GAGAGS hexaamino acid repeat units include about 13% to about 30% alanine, or about 13% alanine, or about 28% alanine, or about 29% alanine, or about 30% alanine, or about 31% alanine. As described herein, proteins such as native silk protein, fibronectin heavy chain, fibronectin light chain, or any protein comprising one or more GAGAGS hexaamino acid repeat units include 9% to about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

In some embodiments, the fibroin-like proteins described herein comprise about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% glycine. In some embodiments, the fibroin-like proteins described herein comprise about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, or about 39% alanine. In some embodiments, the fibroin-like proteins described herein comprise about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, or about 22% serine. In some embodiments, the fibroin-like proteins described herein can independently include any amino acid known to be included in natural fibroin. In some embodiments, the fibroin-like proteins described herein can independently exclude any amino acid known to be included in natural fibroin. In some embodiments, an average of 2 out of 6 amino acids, 3 out of 6 amino acids, or 4 out of 6 amino acids in the fibroin-like proteins described herein are glycine. In some embodiments, an average of 1 out of 6 amino acids, 2 out of 6 amino acids, or 3 out of 6 amino acids in the fibroin-like proteins described herein are alanine. In some embodiments, on average, none of the six amino acids, one of the six amino acids, or two of the six amino acids in the fibronectin-like proteins described herein are serines. Sericin or Sericin Fragments

Raw silk is primarily composed of fibrous protein fibers coated with an adhesive substance called gelatin. Gelatin is a colloidal protein that coats the surface of the silk thread. In addition to glycine and alanine, it is composed of chemically reactive, bulky amino acids such as serine, threonine, and aspartic acid. In the various processes involved in producing silk from raw silk, gelatin plays a crucial role in controlling the solubility of the silk and producing high-quality silk. Furthermore, it plays a crucial role as an adhesive protein. When silk fiber is used as a clothing material, most of the rubber covering the filaments is removed and discarded, making the rubber a valuable unused resource.

In some embodiments, the silk protein fragments described herein include silk or silk fragments. Methods for preparing silk or silk fragments and their applications in various fields are known and described herein, and are also described in, for example, U.S. Patent Nos. 7,115,388, 7,157,273, and 9,187,538, all of which are incorporated herein by reference in their entirety.

In some embodiments, the silk screen removed from the raw silk screen during the debonding step can be collected and used in the methods described herein. Silk screen can also be recovered from the powder and used in the compositions and methods of the present disclosure. Other Properties of SPF

The compositions of the present disclosure are "biocompatible" or otherwise exhibit "biocompatibility," meaning that they are compatible with living tissues or systems because they are non-toxic, non-injurious, or physiologically reactive and do not induce immune rejection or inflammatory responses. Such biocompatibility can be demonstrated by topical application of the compositions of the present disclosure to a subject's skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. For example, in some embodiments, the coating described herein is a biocompatible coating.

In some embodiments, the compositions described herein, which may be biocompatible compositions (e.g., biocompatible coatings comprising filaments), can be evaluated in accordance with the international standard ISO 10993-1, entitled "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein, which may be biocompatible compositions, can be evaluated for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genetic toxicity, carcinogenicity, reproductive and developmental toxicity, and degradation according to ISO 106993-1.

The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by a subject topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

In one embodiment, the stability of the composition of the present disclosure is about 1 day. In one embodiment, the stability of the composition of the present disclosure is about 2 days. In one embodiment, the stability of the composition of the present disclosure is about 3 days. In one embodiment, the stability of the composition of the present disclosure is about 4 days. In one embodiment, the stability of the composition of the present disclosure is about 5 days. In one embodiment, the stability of the composition of the present disclosure is about 6 days. In one embodiment, the stability of the composition of the present disclosure is about 7 days. In one embodiment, the stability of the composition of the present disclosure is about 8 days. In one embodiment, the stability of the composition of the present disclosure is about 9 days. In one embodiment, the stability of the composition of the present disclosure is about 10 days.

In one embodiment, the stability of the disclosed compositions is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days.

In one embodiment, the stability of the composition of the present disclosure is 10 days to 6 months. In one embodiment, the stability of the composition of the present disclosure is 6 months to 12 months. In one embodiment, the stability of the composition of the present disclosure is 12 months to 18 months. In one embodiment, the stability of the composition of the present disclosure is 18 months to 24 months. In one embodiment, the stability of the composition of the present disclosure is 24 months to 30 months. In one embodiment, the stability of the composition of the present disclosure is 30 months to 36 months. In one embodiment, the stability of the composition of the present disclosure is 36 months to 48 months. In one embodiment, the stability of the composition of the present disclosure is 48 months to 60 months.

In one embodiment, the SPF composition of the present disclosure is insoluble in aqueous solution due to the crystallinity of the protein. In one embodiment, the SPF composition of the present disclosure is soluble in aqueous solution. In one embodiment, the SPF of the composition of the present disclosure comprises approximately two-thirds crystalline portion and approximately one-third amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises approximately half crystalline portion and approximately half amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 99% crystalline portion and 1% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 95% crystalline portion and 5% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 90% crystalline portion and 10% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 85% crystalline portion and 15% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 80% crystalline portion and 20% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 75% crystalline portion and 25% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 70% crystalline portion and 30% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 65% crystalline portion and 35% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 60% crystalline portion and 40% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 50% crystalline portion and 50% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 40% crystalline portion and 60% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 35% crystalline portion and 65% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 30% crystalline portion and 70% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 25% crystalline portion and 75% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 20% crystalline portion and 80% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 15% crystalline portion and 85% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 10% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 5% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 1% crystalline portion and 99% amorphous region.

As used herein, the term "substantially free of inorganic residues" means that the composition exhibits 0.1% (w/w) or less residues. In one embodiment, substantially free of inorganic residues means that the composition exhibits 0.05% (w/w) or less residues. In one embodiment, substantially free of inorganic residues means that the composition exhibits 0.01% (w/w) or less residues. In one embodiment, the amount of inorganic residues is between 0 ppm ("non-detectable" or "ND") and 1000 ppm. In one embodiment, the amount of inorganic residues is ND to about 500 ppm. In one embodiment, the amount of inorganic residues is ND to about 400 ppm. In one embodiment, the amount of inorganic residues is ND to about 300 ppm. In one embodiment, the amount of inorganic residues is ND to about 200 ppm. In one embodiment, the amount of inorganic residues is ND to about 100 ppm. In one embodiment, the amount of inorganic residues is between 10 ppm and 1000 ppm.

As used herein, the term "substantially free of organic residues" means that the composition exhibits 0.1% (w/w) or less residues. In one embodiment, substantially free of organic residues means that the composition exhibits 0.05% (w/w) or less residues. In one embodiment, substantially free of organic residues means that the composition exhibits 0.01% (w/w) or less residues. In one embodiment, the amount of organic residues is between 0 ppm ("non-detectable" or "ND") and 1000 ppm. In one embodiment, the amount of organic residues is from ND to about 500 ppm. In one embodiment, the amount of organic residues is ND to about 400 ppm. In one embodiment, the amount of organic residues is ND to about 300 ppm. In one embodiment, the amount of organic residues is ND to about 200 ppm. In one embodiment, the amount of organic residues is ND to about 100 ppm. In one embodiment, the amount of organic residues is between 10 ppm and 1000 ppm.

The compositions of the present disclosure exhibit "biocompatibility," meaning that the compositions are compatible with living tissues or systems because they are non-toxic, non-injurious, or physiologically reactive and do not induce immune rejection. Such biocompatibility can be demonstrated by topical application of the compositions of the present disclosure to the skin of a subject for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by a subject topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

As used herein, in some embodiments, the term "leather" and/or "leather substrate" refers to natural leather, which can be derived from cowhide, sheepskin, lambskin, horsehide, crocodile skin, alligator skin, poultry skin, or another known animal skin as understood in the art, or processed leather. Leather that is unfinished, processed, coated and/or repaired may include, but is not limited to, modified leather, aniline leather, bonded leather, nubuck leather, polished leather, bycast leather, suede leather, chrome-tanned leather, combination-tanned leather, Cordovan leather, corrected grain leather, rubbing leather, tumbled leather, embossed leather, enhanced grain leather, grain leather, metallized leather, bare leather, natural grain leather, nubuck leather, patent leather, pearlized leather, coated leather, printed leather, protected leather, pure aniline leather, tanned/retanned leather, round-handed leather, saddle leather, semi-aniline leather, shrunk grain leather, side leather, split leather, suede leather and wet-blue leather. In some embodiments, the term "leather" may refer to synthetic or reconstituted leather, including but not limited to leather partially/completely composed of cellulose, mushroom-based materials, synthetic materials such as vinyl, synthetic materials such as polyamide, or polyester.

As used herein, the term "hand" refers to the feel of a material, which can be further described as soft, crisp, dry, silky, smooth, and combinations thereof. The hand of a material is also known as "drape." Materials with a hard hand are rough, shaggy, and generally less comfortable to the wearer. Materials with a soft hand are smooth and sleek, and generally more comfortable to the wearer. The hand of a material can be determined by comparison to a collection of material samples, or by using methods such as the Kawabata Evaluation System (KES) or the Fabric Assurance of Simple Tests (FAST) method. Behera and Hari, Ind. J. Fibre & Textile Res., 1994, 19, 168-71. In some embodiments, and as described herein, the silk can change the feel of the leather, as can be assessed by the SynTouch tactile scale method or another method as described herein.

As used herein, "coating" refers to a material or combination of materials that forms a substantially continuous layer or film on the outer surface of a substrate, such as leather or leather products. In some embodiments, a portion of the coating may at least partially penetrate into the substrate. In some embodiments, the coating may at least partially penetrate into the interstices of the substrate. In some embodiments, the coating may be injected into the surface of the substrate, such that the application or coating process of the coating may include at least partially injecting at least one coating component into the surface of the substrate (at the melting temperature of the substrate). The coating may be applied to the substrate by one or more of the processes described herein.

In the described embodiments in which the coating can be injected into the surface of the substrate, the coating can be co-dissolved in the surface of the substrate such that the components of the coating can intermix in the surface of the substrate to a depth of at least about 1 nm, or at least about 2 nm, or at least about 3 nm, or at least about 4 nm, or at least about 5 nm, or at least about 6 nm, or at least about 7 nm, or at least about 8 nm, or at least about 9 nm, or at least about 10 nm, or at least about 20 nm, or at least about 30 nm, or at least about 40 nm, or at least about 50 nm, or at least about 60 nm, or at least about 70 nm, or at least about 80 nm, or at least about 90 nm, or at least about 100 nm. In some embodiments, the coating can be injected into the surface of a substrate, wherein the substrate comprises leather or a leather product.

As used herein, the term "bath coating" encompasses coating a material in a bath, immersing a material in a bath, and submerging a material in a bath. The concept of bath coating is described in U.S. Patent No. 4,521,458, the entire contents of which are incorporated by reference.

As used herein, and unless more specifically described, the term "drying" may refer to drying a coated material as described herein at a temperature above room temperature (ie, 20°C).

Without wishing to be bound by any particular theory, any and all solutions described herein may be further used or processed to obtain a variety of silk and/or SPF compositions, including but not limited to silk non-Newtonian fluids, silk materials that can maintain a shear stress network across the system, silk solutions containing water or another solvent entrapped within a loose silk polymer network, silk solutions converted from liquid form by bond osmosis, Transformed filament materials (such as gels), filaments that entrap mobile solvents to form fixed networks, filament materials that form reversible or irreversible crosslinks, filament materials that exhibit a shear modulus, filament elastomers or filament materials that exhibit thermoplastic behavior, filament materials formed by glass formation, gelation, or colloidal aggregation processes, filament crystals and/or filament crystal polishes, glues, gels, pastes, putties, and/or waxes.

As used herein, when referring to a number or a range of values, the term "about" means that the stated number or range of values is included together with the experimental variability or statistical experimental error of the stated number or range of values, wherein the variability or error is 0% to 15%, or 0% to 10%, or 0% to 5% of the stated number or range of values.

As used herein, "silk-based proteins or fragments thereof" include fibroin-based proteins or fragments thereof, natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof. Natural silk-based proteins or fragments thereof include spider silk-based proteins or fragments thereof, silkworm silk-based proteins or fragments thereof, and combinations thereof. Silkworm silk-based proteins or fragments thereof may include domestic silkworm silk-based proteins or fragments thereof. The SPF mixture solution described herein may include silk-based proteins or fragments thereof. Furthermore, as described herein, SFS may be replaced by the SPF mixture solution. Silk-based proteins or fragments thereof, silk solutions or mixtures (e.g., SPF or SFS solutions or mixtures), and their analogs can be prepared according to the methods described in U.S. Patent Nos. 9,187,538, 9,522,107, 9,522,108, 9,511,012, 9,517,191, and 9,545,369, as well as U.S. Patent Publication Nos. 2016/0222579 and 2016/0281294, and International Patent Publication Nos. WO 2016/090055 and WO 2017/011679, the entire contents of which are incorporated herein by reference. In some embodiments, the silk-based protein or fragment thereof can be provided as a silk composition, which can be an aqueous solution or mixture of silk, silk gel and/or silk wax as described herein.

As used herein, the term "substantially sericin-free" or "substantially devoid of sericin" refers to a silk fiber from which a substantial portion of sericin has been removed. In one embodiment, a silk fiber substantially free of sericin comprises from about 0.01% (w/w) to about 10.0% (w/w) sericin. In another embodiment, a silk fiber substantially devoid of sericin comprises from about 0.01% (w/w) to about 9.0% (w/w) sericin. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 0.01% (w/w) to about 8.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 0.01% (w/w) to about 7.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 0.01% (w/w) to about 6.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk glue refers to a fibrous protein having about 0.01% (w/w) to about 5.0% (w/w) of silk glue. In one embodiment, the fibrous protein substantially free of silk glue refers to a fibrous protein having about 0% (w/w) to about 4.0% (w/w) of silk glue. In one embodiment, the fibrous protein substantially free of silk glue refers to a fibrous protein having about 0.05% (w/w) to about 4.0% (w/w) of silk glue. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 0.1% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 0.5% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 1.0% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 1.5% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 2.0% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein substantially free of silk rubber refers to a fibrous protein having about 2.5% (w/w) to about 4.0% (w/w) of silk rubber. In one embodiment, the fibrous protein that is substantially free of silk glue refers to a fibrous protein having a silk glue content of about 0.01% (w/w) to about 0.1% (w/w). In one embodiment, the fibrous protein that is substantially free of silk glue refers to a fibrous protein having a silk glue content of less than about 0.1% (w/w). In one embodiment, the fibrous protein that is substantially free of silk glue refers to a fibrous protein having a silk glue content of less than about 0.05% (w/w). In one embodiment, when the silk source is added to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes, a degumming loss of about 26 wt% to about 31 wt% is obtained.

As used herein, the term "substantially uniform" may refer to a normal distribution of pure fibrous protein fragments around an identified molecular weight. As used herein, the term "substantially uniform" may refer to a uniform distribution of an additive (e.g., a pigment) throughout the composition of the present disclosure.

As used herein, "residues" refers to materials associated with one or more process steps in the manufacture of a fibroin solution, fibroin fragments, or concentrates thereof.

In some embodiments, the compositions of the present disclosure are "biocompatible" or otherwise exhibit "biocompatibility," meaning that the compositions are compatible with living tissues or systems because they are non-toxic, non-injurious, or physiologically reactive and do not induce immune rejection or inflammatory responses. Such biocompatibility can be demonstrated by topical application of the compositions of the present disclosure to a subject's skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. For example, in some embodiments, the coating described herein is a biocompatible coating.

In some embodiments, the compositions described herein, which in some embodiments may be biocompatible compositions (e.g., biocompatible coatings comprising filaments), can be evaluated in accordance with the international standard ISO 10993-1, entitled "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein, which may be biocompatible compositions, can be evaluated for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genetic toxicity, carcinogenicity, reproductive and developmental toxicity, and degradation according to ISO 106993-1.

In some embodiments, the compositions and articles described herein and methods for preparing the same include yarn-coated leather or leather products. The leather or leather product may be a polymeric material, such as those described elsewhere herein. The terms "infusion" and/or "partial dissolution" include mixing to form a dispersion of, for example, a portion of the leather or leather product and a portion of the silk-based coating. In some embodiments, the dispersion may be a solid suspension of silk (i.e., a dispersion comprising domains on the order of 10 nm) or a solid solution (i.e., a molecular dispersion). In some embodiments, the dispersion may be located at the surface interface between the silk coating and the leather or leather product and may have a depth of 1 nm, 2 nm, 5 nm, 10 nm, 25 nm, 50 nm, 75 nm, 100 nm, or greater than 100 nm, depending on the preparation method. In some embodiments, the dispersion can be a layer sandwiched between the leather or leather product and the silk coating. In some embodiments, the dispersion can be prepared by applying silk (including silk fibroin having the characteristics described herein) to the leather or leather product and then performing an additional process to form the dispersion, the additional process comprising heating at a temperature of 100°C, 125°C, 150°C, 175°C, 200°C, 225°C, or 250°C for a period of time selected from the group consisting of: 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours. In some embodiments, heating can be performed at or above the glass transition temperature ( _Tg ) of the silk and/or polymer fabric or textile, which can be assessed by methods known in the art. In some embodiments, the dispersion can be formed by coating silk (including silk fibroin having the characteristics described herein) onto leather or leather products, and then performing an additional process to impregnate the silk coating into the leather or leather products, the additional process including treatment with an organic solvent. Methods for characterizing the properties of polymers that are soluble in each other are well known in the art and include differential scanning calorimetry and surface analysis methods capable of depth profiling, including spectroscopy.

In some embodiments, the compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by a subject applying the compositions of the present disclosure topically to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

In some embodiments, when an aqueous solution is used to prepare an SPF composition or an SPF-containing coating, any type of water is used to prepare the aqueous solution. In some embodiments, the water may be deionized water, tap water, or naturally available water. As used herein, "tap water" refers to drinking water provided by a public utility and water of comparable quality, regardless of source, without further refinement, such as by reverse osmosis, distillation, and/or deionization. Therefore, the use of "DI water," "RODI water," or "water" as described herein is understood to be interchangeable with "tap water" in accordance with the processes described herein without adversely affecting such processes. Leather and leather products processed, coated, and/or repaired with fibrous protein-based protein fragments

In one aspect, the present disclosure provides a coating composition comprising fibroin or a fragment thereof. In one embodiment, the fibroin or fragment thereof has an average weight average molecular weight in a range selected from about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, and has a polydispersity between 1 and about 5. In some embodiments, the fibrous protein or its fragment has any average weight average molecular weight described herein. In some embodiments, the polydispersity of the fibrous protein or its fragment is between 1 and about 1.5. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 1.5 and about 2. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 2 and about 2.5. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 2.5 and about 3. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 3 and about 3.5. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 3.5 and about 4. In some embodiments, according to technical solution 1, the polydispersity of the fibrous protein or its fragment is between about 4 and about 4.5. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 4.5 and about 5.

In one embodiment, the fibrous protein or fragment thereof has any of the weight average molecular weight and polydispersity described herein, and optionally any other limitations described herein, and the silk gel is from about 0.001% (w/w) to about 10% (w/w) relative to the fibrous protein or fragment thereof. In some embodiments, the w/w ratio between fibroin or a fragment thereof and sericin is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, about 76:24, or about 75:25. In some embodiments, the relative w/w amount of silk glue and silk fibroin or its fragments is about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.01% or about 0.001%.

In one embodiment, the silk fibroin or fragment thereof has any of the weight average molecular weight and polydispersity described herein, and optionally any other limitations described herein, wherein the silk fibroin or fragment thereof does not gel spontaneously or gradually and does not significantly change in color or turbidity when in aqueous solution for at least 10 days before being applied to an article. In some embodiments, the fibrous protein or fragment thereof does not spontaneously or gradually gel and does not significantly change color or turbidity when in aqueous solution for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 4 weeks, or 1 month before being applied to an article.

In one aspect, the present disclosure provides an article coated with a coating composition described elsewhere herein.

In one embodiment, the article is a leather article, such as a leather substrate. Some methods for adding protein to substrates (including leather substrates) are described in U.S. Patent No. 8,993,065, which is incorporated herein by reference in its entirety. The present disclosure also provides an article of manufacture comprising a leather substrate and fibrous protein or a fragment thereof, wherein the fibrous protein or a fragment thereof has any of the weight average molecular weight and polydispersity described herein, and optionally any other limitations described herein, wherein: 1) a portion of the fibrous protein or a fragment thereof is coated on the surface of the leather substrate; or 2) a portion of the fibrous protein or a fragment thereof is impregnated into a layer of the leather substrate, in some embodiments, such layer having a thickness as described herein; or 3) a portion of the fibrous protein or a fragment thereof is located in a recessed portion of the leather substrate, the recessed portion being selected from openings, crevices, and defects in the leather substrate; or 4) any combination of the foregoing.

With reference to Figures 22A and 22B, the manner in which a portion of a fibrous protein or a fragment thereof is coated on the surface of a leather substrate, or the manner in which a portion of a fibrous protein or a fragment thereof is located in a recessed portion of a leather substrate, can be described by a cross-sectional index, wherein the cross-sectional index is defined as the ratio between the area above the curve up to the baseline and the length of the cross-section spanned by the area above the defined curve. The cross-sectional index is herein reflected as a unitless value. The curve may reflect the leather surface along the cross-sectional surface (if uncoated or unfilled), or the surface of the fibrous protein or a fragment thereof coated or filled along the cross-sectional surface. The baseline may reflect a horizontal plane close to the surface of the leather substrate, through which the cross-sectional index is determined.

As shown in FIG49A , for example, the recessed portion is between a cross-sectional area x1 = about 210 μm and a cross-sectional area x2 = about 600 μm, and the cross-sectional index of this recessed portion can be calculated as described herein. In some embodiments, the recessed portion of the leather substrate has a cross-sectional index of about 6.50, about 6.75, about 7, about 7.25, about 7.50, about 7.75, about 8, about 8.25, about 8.50, about 8.75, about 9, about 9.25, about 9.50, about 9.75, or about 10. In some embodiments, the recessed portion of the leather substrate may have another cross-sectional index, such as about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7. 2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10. As also shown in FIG. 49A , the substantially non-recessed portion of the leather substrate is, for example, between the cross-sectional area x1 = 0 μm and x2 = about 210 μm, and the cross-sectional index of this substantially non-recessed portion can be calculated as described herein. In some embodiments, the substantially non-recessed portion of the leather substrate has a cross-sectional index of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0. In some embodiments, the substantially non-recessed portion of the leather substrate may have another cross-sectional index, such as about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.

As shown in FIG22B , the concave portion filled with fibroin or a fragment thereof has a cross-sectional area x1 = approximately 210 μm and x2 = approximately 395 μm, and the cross-sectional index of the filled concave portion can be calculated as described herein. In some embodiments, the filled concave portion of the leather substrate can have a cross-sectional index of about 0.25, about 0.50, about 0.75, about 1, about 1.25, about 1.27, about 1.50, about 1.75, or about 2. In some embodiments, the filled recessed portion of the leather substrate may have any other cross-sectional index, such as about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3. As also shown in FIG22B , the substantially non-recessed portion of the leather substrate coated with fibroin or a fragment thereof is, for example, between the cross-sectional area x1 = 0 μm and x2 = approximately 210 μm, and the cross-sectional index of this recessed portion can be calculated as described herein. In some embodiments, the coated substantially non-recessed portion of the leather substrate has a cross-sectional index of about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.50, about 0.75, about 1, about 1.25, about 1.27, about 1.50, about 1.75, or about 2. In some embodiments, the coated substantially non-recessed portion of the leather substrate may have any other cross-sectional index, such as about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.

In some embodiments, the coated, substantially non-recessed portion of the leather substrate may have a lower cross-sectional index than the substantially non-recessed portion of the leather substrate before coating. In some embodiments, the coated, substantially non-recessed portion of the leather substrate has a lower cross-sectional index than the substantially non-recessed portion of the leather substrate before coating, wherein the cross-sectional index of the coated, substantially non-recessed portion of the leather substrate is greater than 0. In some embodiments, the cross-sectional index of the coated, substantially non-recessed portion of the leather substrate is lower by a factor of 1% to 99% than the substantially non-recessed portion of the leather substrate before coating.

In some embodiments, the coated, substantially non-recessed portion of the leather substrate may have a lower cross-sectional index than the substantially recessed portion of the leather substrate before filling. In some embodiments, the coated, substantially non-recessed portion of the leather substrate has a lower cross-sectional index than the substantially recessed portion of the leather substrate before filling, wherein the cross-sectional index of the coated, substantially non-recessed portion of the leather substrate is higher than 0. In some embodiments, the cross-sectional index of the coated, substantially non-recessed portion of the leather substrate is lower by a factor of 1% to 99% than the substantially recessed portion of the leather substrate before filling.

In some embodiments, the filled recessed portions of the leather substrate may have a lower cross-sectional index than the substantially non-recessed portions of the leather substrate before coating. In some embodiments, the filled recessed portions of the leather substrate may have a lower cross-sectional index than the substantially non-recessed portions of the leather substrate before coating, wherein the cross-sectional index of the filled recessed portions of the leather substrate is higher than 0. In some embodiments, the cross-sectional index of the filled recessed portions of the leather substrate may be lower by a factor of 1% to 99% than that of the substantially non-recessed portions of the leather substrate before coating.

In some embodiments, the filled recessed portion of the leather substrate may have a lower cross-sectional index than the substantially non-recessed portion of the leather substrate before filling. In some embodiments, the filled recessed portion of the leather substrate may have a lower cross-sectional index than the substantially non-recessed portion of the leather substrate before filling, wherein the cross-sectional index of the filled recessed portion of the leather substrate is higher than 0. In some embodiments, the cross-sectional index of the filled recessed portion of the leather substrate may be lower by a factor of 1% to 99% than the substantially non-recessed portion of the leather substrate before filling.

The present disclosure also provides an article comprising a leather substrate and fibrous proteins or fragments thereof, wherein the fibrous proteins or fragments thereof have any weight-average molecular weight and polydispersity as described herein, and optionally any other limitations as described herein, the article further comprising one or more polysaccharides selected from the group consisting of starch, cellulose, gum arabic, guar gum, xanthan gum, alginate, pectin, chitin, chitosan, carrageenan, inulin, and gellan gum. In some embodiments, the polysaccharide is gellan gum. In some embodiments, the gellan gum comprises gellan gum having a low acyl content.

The present disclosure also provides an article comprising a leather substrate and a fibrous protein or a fragment thereof, wherein the fibrous protein or the fragment thereof has any weight average molecular weight and polydispersity as described herein, and optionally any other limitations as described herein, the article further comprising one or more polyols and/or one or more polyethers. In some embodiments, the polyol comprises one or more of a glycol, glycerol, sorbitol, glucose, sucrose, and dextrose.

The present disclosure also provides an article comprising a leather substrate and fibrous proteins or fragments thereof, wherein the fibrous proteins or fragments thereof have any of the weight average molecular weights and polydispersities described herein, and optionally any other limitations described herein, the article further comprising one or more of a polysilicone, a dye, a pigment, and a polyurethane as described herein.

In one embodiment, the water-based coating composition described elsewhere herein is applied directly to an article. In one embodiment, a silk coating layer described elsewhere herein can be applied to an article to form a pattern or design on the article. In one embodiment, the coating composition described elsewhere herein is applied to leather or leather products under tension and/or relaxation to modify penetration into the leather or leather products. In one embodiment, the present disclosure provides leather and leather products coated with the silk composition described herein. In one embodiment, the present disclosure provides leather and leather products repaired with the silk composition described herein, for example, by filling, masking, or concealing defects in the surface or structure of the leather.

In one embodiment, the present disclosure provides leather and leather products processed with any of the silk compositions and dyes described herein to provide colored leather and leather products exhibiting enhanced color saturation and excellent color fixation properties. In some embodiments, the silk composition can be applied simultaneously with the dye. In some embodiments, the silk composition can be applied before the dyeing process. In some embodiments, the silk composition can be applied after the dyeing process. In some embodiments, the leather can include crust nubuck leather, nubuck leather finished in black or blue, suede leather finished in brown or turquoise, bottom split suede leather, or top split wet blue suede leather.

The present disclosure generally provides methods and articles involving filling recessed portions of leather, such as, but not limited to, openings, gaps, or defects in a leather substrate, with silk fibroin and/or fragments thereof. As used herein, the term "defect" or "leather defect" refers to any defect in the surface of the leather or in the surface and/or underlying structure. For example, the removal of hair and/or hair follicles may leave visible voids or gaps in the surface or structure of a leather or hide. The present disclosure is not limited to repairing visible defects, and therefore, it is contemplated that any defect may be repaired as described herein. The present disclosure is also not limited to repairing defects of a particular size, and defects of any size may be repaired and/or filled. For example, silk and/or SPF and any and all compositions described herein may be used to fill or mask the appearance of larger defects that appear over a larger area of a defective skin surface.

As used herein, "repaired" or "repaired" leather refers to leather that has had a defect filled with a composition comprising silk and/or SPF, wherein the defect is substantially eliminated as a result of such repair. For example, a void or gap that is fully or partially filled with a composition as described herein may be a repaired defect.

In one embodiment, the present disclosure provides leather or leather products processed, coated and/or repaired with a fibrous protein or a fragment thereof. In one embodiment, the present disclosure provides leather or leather products processed, coated or repaired with a fibrous protein or a fragment thereof, wherein the leather or leather products are leather or leather products for human clothing (including clothing). In one embodiment, the present disclosure provides leather or leather products processed, coated or repaired with a fibrous protein or a fragment thereof, wherein the leather or leather products are used for automotive interiors. In one embodiment, the present disclosure provides leather or leather products processed, coated or repaired with a fibrous protein or a fragment thereof, wherein the leather or leather products are used for aircraft interiors. In one embodiment, the present disclosure provides leather or leather products processed, coated, or repaired with a fibrous protein or a fragment thereof, wherein the leather or leather products are used for interior trim in transportation vehicles (including buses and trains) for public, commercial, military, or other purposes. In another embodiment, the present disclosure provides leather or leather products processed, coated, or repaired with a fibrous protein or a fragment thereof, wherein the leather or leather products are used for interior trim in products requiring a high degree of wear resistance compared to normal interior trim.

In one embodiment, the leather or leather product is treated with a polymer such as polyglycolide (PGA), polyethylene glycol, copolymers of glycolide, glycolide/L-lactide copolymers (PGA/PLLA), glycolide/trimethylene carbonate copolymers (PGA/TMC), polylactide (PLA), stereocopolymers of PLA, poly-L-lactide (PLLA), poly-DL-lactide (PDLLA), L-lactide/DL-lactide copolymers, copolymers of PLA, lactide/tetramethyl glycolide copolymers, lactide/trimethylene carbonate copolymers, lactide/δ-valerolactone copolymers, lactide/ε-caprolactone copolymers, polyphenol peptides, PLA/polyethylene oxide copolymers, asymmetric 3,6-substituted poly-1,4-diol alkane-2,5-diones, poly-β-hydroxybutyrate (PHBA), PHBA/β-hydroxyvalerate copolymer (PHBA/HVA), poly-β-hydroxypropionate (PHPA), poly(p-dioxanone) (PDS), poly-δ-valerolactone, poly-ε-caprolactone, methyl methacrylate-N-vinylpyrrolidine copolymer, polyesteramide, polyester oxalate, polydihydropyran, polyalkyl-2-cyanoacrylate, polyurethane (PU), polyvinyl alcohol (PVA), polypeptide, poly-β-malic acid (PMLA), poly-β-alkanoic acid, polyvinyl alcohol (PVA), polyethylene oxide (PEO), chitin polymers, polyethylene, polypropylene, polyacetal, polyamide, polyester, polysulfone, polyetheretherketone, polyethylene terephthalate, polycarbonate, polyaryletherketone, and polyetherketoneketone.

In one embodiment, an aqueous solution of the pure silk fibroin-based protein fragments disclosed herein is used to process and/or coat leather or leather products. In one embodiment, the silk concentration in the solution ranges from about 0.1% to about 20.0%. In one embodiment, the silk concentration in the solution ranges from about 0.1% to about 15.0%. In one embodiment, the silk concentration in the solution ranges from about 0.5% to about 10.0%. In one embodiment, the silk concentration in the solution ranges from about 1.0% to about 5.0%. In one embodiment, the aqueous solution of the pure silk fibroin-based protein fragments disclosed herein is applied directly to leather or leather products. Alternatively, the silk microspheres and any additives can be used to process and/or coat leather or leather products. In one embodiment, an additive may be added to the aqueous solution of the pure silk fibroin-based protein fragment disclosed herein before coating (e.g., alcohol) to further enhance the material properties. In one embodiment, the silk coating disclosed herein may have

In one embodiment, the composition of pure fibrous protein-based protein fragments disclosed herein is used to repair leather or leather products. In some embodiments, the composition is viscous. In some embodiments, the composition is thixotropic. In some embodiments, the composition is a gel, putty, wax, paste, or the like. In some embodiments, the composition is formed into a repair stick, such as a repair crayon. In some embodiments, the composition is delivered from a syringe, a delivery gun, a brush-type applicator, a roller-type applicator, a pen or marker-type applicator, or the like. In some embodiments, the composition is delivered from multiple syringes (e.g., a dual syringe or dual delivery gun) along with different compositions designed to harden the SPF composition, trigger curing of the SPF composition, or otherwise modify the SPF composition. In one embodiment, the silk concentration in the composition ranges from about 0.1% to about 50.0%. In one embodiment, the silk concentration in the solution ranges from about 0.1% to about 35.0%. In one embodiment, the silk concentration in the solution ranges from about 0.5% to about 30.0%. In one embodiment, the silk concentration in the solution ranges from about 1.0% to about 25.0%. In one embodiment, the composition of pure silk fibroin-based protein fragments disclosed herein is applied directly to leather or leather products, such as leather defects. Alternatively, the silk microspheres and any additives can be used to repair leather or leather products. In one embodiment, an additive may be added to the composition of the pure filamentous protein-based protein fragment disclosed herein before coating (e.g., alcohol) to further enhance the material properties. In one embodiment, the composition is applied to leather or leather products under tension and/or relaxation to alter penetration into the leather, leather products, or leather defects. In one embodiment, the disclosure provides leather or leather products coated with a coating composition described elsewhere herein. In one embodiment, the leather or leather product is aniline leather or leather products. In one embodiment, the leather or leather product is used for human clothing, automotive interiors, aircraft interiors, or interiors in transportation vehicles (including buses and trains) for public, commercial, military, or other purposes. In one embodiment, the present disclosure provides leather or leather products coated with a matte coating composition described elsewhere herein, wherein the leather or leather product is used in products requiring a matte effect. In another embodiment, the present disclosure provides aniline leather or aniline leather products coated with a water-soluble dye-fixing coating composition described elsewhere herein. In one embodiment, the aniline leather or aniline leather product is coated with a water-soluble dye-fixing coating composition comprising a water-soluble aniline leather dye as described herein. In one embodiment, the water-soluble dye-fixing coating composition comprising a water-soluble aniline leather dye as described herein fixes the aniline leather dye during the finishing of the aniline leather, thereby providing a dyed leather with a natural look and/or feel. In one embodiment, the concentration of the water-soluble dye and/or water-soluble aniline leather dye can be adjusted to provide a darker color for the dyed leather or a lighter color for the dyed leather. Method for coating an article with a coating composition

In yet another aspect, the present disclosure provides a method of coating an article with a coating composition, the method comprising applying the coating composition to one or more surfaces of the article.

Coating compositions are described elsewhere herein. In one embodiment, the coating composition comprises fibrous protein or a fragment thereof and matte silica and/or starch. In another embodiment, the coating composition comprises fibrous protein or a fragment thereof and a water-soluble dye. In another embodiment, the coating composition is a two-component coating composition, wherein the first component comprises the water-soluble dye and the second component comprises fibrous protein or a fragment thereof.

In one embodiment, the coating composition is a liquid, gel, paste, wax, or cream. In one embodiment, the coating composition is a liquid. In one embodiment, the coating composition comprises an aqueous solvent. In one embodiment wherein the coating composition is a liquid, the method further comprises the step of drying the product.

The coating compositions described herein can be applied to an article using any method known to those skilled in the art. Exemplary application methods include, but are not limited to, manual spraying, spraying using a mechanical spraying device, application by brushing, rubbing, wet mixing, washing, drumming, soaking, injection, plastering, painting, or the like. In one embodiment wherein the coating composition comprises fibrous protein or a fragment thereof and a water-soluble dye, the coating composition is sprayed onto the article in a single application of approximately 4 g/sqft. In one embodiment, the coating composition comprising fibrous protein or a fragment thereof and a water-soluble dye is sprayed onto a leather article in a single application of approximately 4 g/sqft.

In some embodiments, the coating compositions described herein can be applied to an article alone, mixed with one or more chemicals (e.g., chemicals), as a single coating layer, multiple coating layers, or multiple times using different application methods. In one embodiment, the coating layer has a thickness as described elsewhere herein.

In embodiments where the coating composition is a two-component coating composition, the step of applying the coating composition to one or more surfaces of an article comprises (a) applying a first portion of the coating composition to one or more surfaces of the article. In one embodiment, the first portion of the coating composition comprising a water-soluble dye is applied to one or more surfaces of the article. In one embodiment, the first portion of the coating composition comprising a water-soluble dye is applied to one or more surfaces of a leather article. In one embodiment, the first portion of the coating composition is applied to the leather article by spraying. In one embodiment, the first portion of the coating composition is applied to the leather article by spraying a first layer onto the article at approximately 2 g/sqft.

In one embodiment, the method further comprises the step (b) of drying the leather article after applying the first layer of the first part of the coating composition. In one embodiment, the method further comprises (c) applying a second layer of the first part of the coating composition to one or more surfaces of the dried leather article. In one embodiment, the first part of the coating composition is applied to the dried leather article by spraying. In one embodiment, the first part of the coating composition is applied to the dried leather article by spraying the second layer onto the article at approximately 2 g/sqft.

In some embodiments, the method further comprises (d) drying the leather article after applying the second layer of the first part of the coating composition to the article.

In one embodiment, the method further comprises (e) applying a second portion of the coating composition to one or more surfaces of the article. In one embodiment, the second portion of the coating composition comprising fibrous protein or a fragment thereof is applied to one or more surfaces of the article. In one embodiment, the second portion of the coating composition comprising fibrous protein or a fragment thereof is applied to one or more surfaces of a leather article. In one embodiment, the second portion of the coating composition is applied to the leather article by spraying. In one embodiment, the second portion of the coating composition is applied to the leather article by spraying a layer of the second portion of the coating composition onto the article at a rate of approximately 4 g/sqft. In one embodiment, only one layer of the second part of the coating composition is applied to the leather article.

In one embodiment, the method further comprises (f) drying the leather article after applying the second part of the coating composition to the article.

In one embodiment, a coating composition comprising fibrous protein or a fragment thereof and a water-soluble dye exhibits comparable performance as a two-component coating composition when applied as a coating to an article. In one embodiment, the one-component coating composition exhibits similar colorfastness to rubbing as the two-component coating composition. Leather Products

In one embodiment, the present disclosure provides a method for preparing leather and leather products coated or repaired with the coating composition described herein. In one embodiment, the coating composition comprises fibroin or a fragment thereof and matte silica and/or starch.

As shown in Figure 3, the following steps can be used in the leather preparation process: ●    Unhairing - soaking the skin in an alkaline solution to remove hair; ●    Liming - soaking the skin in an alkaline/sulfide solution to change the properties of the collagen, causing it to swell and take on a more open structure; ●    Deliming and bating - an enzymatic treatment that further opens up the skin's collagen structure; ●    Pickling - an acid treatment that preserves the skin; ●    Tanning - a chemical process in which some of the bound collagen structure is replaced by complex ions of chromium (wet blue leather); ●  Neutralization, dyeing, and fatliquoring—alkaline neutralization solutions prevent deterioration, apply various compounds, and react at chromium-active sites, including oils that attach themselves to collagen fibers; ● Drying—removes water and stabilizes the leather's chemical properties; and ● Finishing—applies a surface coating to ensure uniform color and texture. Mechanical treatments can be performed before or after the finishing process to adjust material characteristics/solidify the chemistry.

The present disclosure provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to a surface of the leather, the silk formulation comprising silk fibroin or a fragment thereof having an average weight average molecular weight in a range selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa. kDa, and a polydispersity between 1 and about 5. In some embodiments, any other average weight average molecular weight and polydispersity described herein may be used. In some embodiments, the polydispersity of the fibrous protein or its fragments is between 1 and about 1.5. In some embodiments, the polydispersity of the fibrous protein or its fragments is between about 1.5 and about 2. In some embodiments, the polydispersity of the fibrous protein or its fragments is between about 2 and about 2.5. In some embodiments, the polydispersity of the fibrous protein or its fragments is between about 2.5 and about 3. In some embodiments, the polydispersity of the fibrous protein or its fragments is between about 3 and about 3.5. In some embodiments, the polydispersity of the fibrous protein or its fragments is between about 3.5 and about 4. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 4 and about 4.5. In some embodiments, the polydispersity of the fibrous protein or its fragment is between about 4.5 and about 5.

The present disclosure provides a method for coating a leather substrate with a coating composition, the method comprising applying the coating composition to one or more surfaces of the leather substrate.

In some embodiments, a method for treating a leather substrate with a silk formulation comprises applying a silk formulation to the surface of the leather, the silk formulation comprising silk fibroin or a fragment thereof having any of the average weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk formulation further comprises about 0.001% (w/w) to about 10% (w/w) of silk glue relative to the silk fibroin or a fragment thereof. In some embodiments, the w/w ratio between fibroin or a fragment thereof and sericin is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, about 76:24, or about 75:25. In some embodiments, the relative w/w amount of silk glue and silk fibroin or its fragments is about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.01% or about 0.001%.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any of the weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk formulation further comprises about 0.001% (w/v) to about 10% (w/v) of silk glue. In some embodiments, the silk formulation further comprises about 0.001% (w/v) of silk to about 0.01% (w/v) of silk, about 0.01% (w/v) of silk to about 0.1% (w/v) of silk, about 0.1% (w/v) of silk to about 1% (w/v) of silk, or about 1% (w/v) of silk to about 10% (w/v) of silk. In some embodiments, the silk formulation further comprises about 1% (w/v) of silk, about 2% (w/v) of silk, about 3% (w/v) of silk, about 4% (w/v) of silk, about 5% (w/v) of silk, about 6% (w/v) of silk, about 7% (w/v) of silk, about 8% (w/v) of silk, about 9% (w/v) of silk, about 10% (w/v) of silk, about 11% (w/v) of silk, about 12% (w/v) of silk, about 13% (w/v) of silk, about 14% (w/v) of silk, about 16% (w/v) of silk, about 17% (w/v) of silk, about 18% (w/v) of silk, about 19% (w/v) of silk, about 20% (w/v) of silk, about 21% (w/v) of silk, about 22% (w/v) of silk, about 23% (w/v) of silk, about 24% (w/v) of silk, about 25% (w/v) of silk, about 26% (w/v) of silk, about 27% (w/v) of silk, about 28% (w/v) of silk, about 29% (w/v) of silk, about 30% (w/v) of silk, about 31% (w/v) of silk, about 32% (w/v) of silk, about 33% (w/v) of silk, about 34% (w/v) of silk, about 35% (w/v) of silk, about 36% (w/v) of silk, about 37% (w/v) of silk, about 38% (w/v) of silk, about 39% (w/v) of silk, about 40% (w/v) of silk, about 41 (w/v) of silk, about 12% (w/v) of silk, about 12% (w/v) of silk, about 13% (w/v) of silk, about 14% (w/v) of silk, or about 15% (w/v) of silk.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying to the surface of the leather a silk formulation comprising a silk fibroin or fragment thereof having any of the weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk fibroin or fragment thereof does not spontaneously or gradually gel and does not significantly change color or turbidity when in aqueous solution for at least 10 days prior to formulation and application to the leather substrate. In some embodiments, the fibrous protein or fragment thereof does not spontaneously or gradually gel and does not significantly change in color or turbidity in aqueous solution for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 4 weeks, or 1 month.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying to the surface of the leather a silk formulation comprising a silk fibroin or fragment thereof having any of the weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk fibroin or fragment thereof does not spontaneously or gradually gel and does not significantly change color or turbidity when in the formulation for at least 10 days prior to application to the leather substrate. In some embodiments, the fibroin or fragment thereof does not spontaneously or gradually gel and does not noticeably change color or turbidity in the formulation for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 4 weeks, or 1 month.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation comprising a silk fibroin or a fragment thereof having any of the weight-average molecular weights and polydispersities described herein to the surface of the leather, and optionally any other steps described herein, wherein in some embodiments: 1) a portion of the silk formulation is applied to the surface of the leather substrate; or 2) a portion of the silk formulation is injected into a layer of the leather substrate; or 3) a portion of the silk formulation is introduced into a recessed portion of the leather substrate, the recessed portion being selected from an opening, a crevices, and a defect in the leather substrate; or 4) any combination thereof. The silk formulation can be applied to any desired thickness, for example, but not limited to, from about 1 μm to about 100 μm. In some embodiments, coating thickness refers to wet coating. In some embodiments, coating thickness refers to dried coating thickness. The silk formulation can be injected into a substrate layer having any thickness, such as, but not limited to, from about 1 µm to about 100 µm. In some embodiments, injection layer thickness refers to wet injection. In some embodiments, injection layer thickness refers to injection after drying.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any weight average molecular weight and polydispersity as described herein, and optionally any other steps as described herein, wherein in some embodiments, the silk formulation further comprises a rheology modifier. In some embodiments, the rheology modifier comprises one or more polysaccharides, including one or more of starch, cellulose, gum arabic, guar gum, xanthan gum, alginate, pectin, chitin, chitosan, carrageenan, inulin and/or gellan gum. In some embodiments, the polysaccharide comprises gellan gum, including but not limited to gellan gum with a low acyl content. In some embodiments, the w/w ratio of the silk fibroin or its fragments to the rheology modifier in the silk formulation is about 25:1 to about 1:5. In some embodiments, the w/w ratio of the silk fibroin or its fragments to the rheology modifier in the silk formulation is about 12:1 to about 0.1:1. In some embodiments, the w/w ratio of the silk fibroin or its fragments to the rheology modifier in the silk formulation is about 99:1 to about 1:99. In some embodiments, the w/v concentration of the rheology modifier in the silk formulation is about 0.01% to about 5%. In some embodiments, the w/v concentration of the rheology modifier in the silk formulation is about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.01%, or about 0.001%. In some embodiments, the w/v concentration of the rheology modifier in the silk formulation is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation on the surface of the leather, the silk formulation comprising a silk fibroin or its fragment having any average weight average molecular weight and polydispersity described herein, and any other steps described herein as appropriate, wherein in some embodiments, the silk formulation further comprises a plasticizer. In some embodiments, the plasticizer comprises one or more polyols and/or one or more polyethers. In some embodiments, the polyol is selected from one or more of glycol, glycerol, sorbitol, glucose, sucrose, and dextrose. In some embodiments, the polyether is one or more polyethylene glycols (PEGs). In some embodiments, the w/w ratio between the silk fibroin or its fragment and the plasticizer in the silk formulation is about 5:1 to about 1:5. In some embodiments, the w/w ratio of silk fibroin or a fragment thereof to the plasticizer in the silk formulation is about 99:1 to about 1:99. In some embodiments, the w/v concentration of the plasticizer in the silk formulation is about 0.01% to about 10%. In some embodiments, the w/v concentration of the plasticizer in the silk formulation is about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.01%, or about 0.001%. In some embodiments, the w/v concentration of the plasticizer in the silk formulation is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any of the weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk formulation further comprises a defoaming agent at a concentration of about 0.001% to about 1%, about 0.01% to about 2.5%, about 0.1% to about 3%, about 0.5% to about 5%, or about 0.75% to about 7.5%. In some embodiments, the defoaming agent comprises silicone. The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any of the weight-average molecular weights and polydispersities described herein, and optionally any other steps described herein, wherein in some embodiments, the silk formulation further comprises a degassing agent at a concentration of about 0.001% to about 1%, about 0.01% to about 2.5%, about 0.1% to about 3%, about 0.5% to about 5%, or about 0.75% to about 7.5%. In some embodiments, the degassing agent comprises silicone.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation comprising a silk fibroin or a fragment thereof having any of the weight-average molecular weights and polydispersities described herein to the surface of the leather, and optionally any other steps described herein, wherein in some embodiments, the silk formulation is a liquid, a gel, a paste, a wax, or a cream.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any of the weight-average molecular weights and polydispersities described herein, and optionally any other steps described herein, wherein in some embodiments, the concentration of the silk fibroin or a fragment thereof in the silk formulation is from about 0.1% w/v to about 15% w/v. In some embodiments, the concentration of the silk fibroin or a fragment thereof in the silk formulation is from about 0.5% w/v to about 12% w/v. In some embodiments, the concentration of silk fibroin or a fragment thereof in the silk formulation is about 1% w/v, about 1.5% w/v, about 2% w/v, about 2.5% w/v, about 3% w/v, about 3.5% w/v, about 4% w/v, about 4.5% w/v, about 5% w/v, about 5.5% w/v, about 6% w/v, about 6.5% w/v, about 7% w/v, about 7.5% w/v, about 8% w/v, about 8.5% w/v, about 9% w/v, about 9.5% w/v, or about 10% w/v. In some embodiments, the concentration of the silk fibroin or its fragment in the silk formulation is about 3% w/v, about 3.25% w/v, about 3.5% w/v, about 3.75% w/v, about 4% w/v, about 4.25% w/v, about 4.5% w/v, about 4.75% w/v, about 5% w/v, about 5.25% w/v, about 5.5% w/v, about 5.75% w/v, about 6% w/v, about 6.25% w/v, about 6.5% w/v, about 6.75% w/v, about 7% w/v, about 7.25% w/v, about 7.5% w/v, about 7.75% w/v, about 8% w/v, about 8.25% w/v, about 8.5% w/v, about 8.75% w/v w/v, about 9% w/v, about 9.25% w/v, about 9.5% w/v, about 9.75% w/v, or about 10% w/v. In some embodiments, the concentration of the silk fibroin or its fragment in the silk formulation is about 5 mg/mL to about 125 mg/mL. In some embodiments, the concentration of the silk fibroin or its fragment in the silk formulation is about 10 mg/mL to about 110 mg/mL.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or fragment thereof having any average weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, the silk formulation further comprises a pH adjuster. In some embodiments, the pH adjuster comprises one or more of an acid and/or a base, including but not limited to a weak acid and/or a weak base. In some embodiments, the pH adjuster comprises one or more of ammonium hydroxide and citric acid. Any hydroxide or weak carboxylic acid can be used interchangeably with any of the above. In some embodiments, the pH of the silk formulation is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.

The present disclosure also provides a method for treating a leather substrate with a silk formulation, the method comprising applying a silk formulation to the surface of the leather, the silk formulation comprising a silk fibroin or a fragment thereof having any of the weight average molecular weight and polydispersity described herein, and optionally any other steps described herein, wherein in some embodiments, treating the leather substrate with the silk formulation improves one or more of gloss and/or color saturation and/or smoothness.

In some embodiments, the method further comprises one or more additional steps, such as dyeing the leather substrate, drying the leather substrate, mechanically stretching the leather substrate, trimming the leather substrate, performing one or more polishing steps on the leather substrate, applying a pigment to the leather substrate, applying a colorant to the leather substrate, applying an acrylic formulation to the leather substrate, chemically fixing the leather substrate, stamping the leather substrate, applying a silicone coating to the leather substrate, providing the leather substrate with a Uniflex treatment, and/or providing the leather substrate with a Finiflex treatment, wherein the step of applying the coating composition to one or more of the leather substrate is performed before, during, or after the one or more additional steps.

As described herein, the silk and/or SPF compositions described herein can be used before, during, or after any of these steps. In some embodiments, the leather preparation process can include treating leather with the silk compositions described herein. In some embodiments, the leather preparation process can include repairing leather with the silk compositions described herein. In some embodiments, the silk composition can include one or more chemicals (e.g., silicone, polyurethane, etc.) as described below.

In some embodiments, the thread is applied by hand spraying, spraying using a mechanical spraying device, by brushing, rubbing, wet mixing, washing, drumming, dipping, injecting, plastering, painting, or the like.

In some embodiments, the silk compositions described herein can be applied alone, mixed with one or more chemicals (e.g., chemicals), as a single coat, multiple coats, or as a defect filling composition, using various application methods to leather that has or has not been dyed, chromed, or sprayed with pigments, acrylics, fixatives, finishes, and/or colorants. In some embodiments, the silk compositions can also be applied by hand spraying, spraying using a mechanical spraying device, by brushing, rubbing, wet mixing, washing, drumming, dipping, injecting, plastering, painting, or the like.

In some embodiments, the silk compositions described herein can be applied alone, mixed with one or more chemicals (e.g., chemicals), as a single coat, multiple coats, or as a defect filling composition, using different application methods to leather that has or has not been dyed, chromed, or sprayed with pigments, acrylics, fixatives, finishes, and/or colorants.

The coating compositions described herein can be applied to leather or leather products by any method described herein. In some embodiments, the coating compositions described herein can be applied to finished leather or leather products, mechanically treated leather or leather products, or drum-processed leather or leather products.

In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the liming step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the deliming and/or bating step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the pickling step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the tanning step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the neutralization, dyeing, and/or fatliquoring steps. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after the drying step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after any finishing step.

In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the liming step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the deliming and/or bating steps. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the pickling step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the tanning step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the neutralization, dyeing, and/or fatliquoring steps. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the drying step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather during the finishing step. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used during or as part of the finishing step.

In one embodiment, the coating compositions described herein (with or without one or more chemicals) can be used during or as part of a coating step. In another embodiment, the coating compositions described herein (with or without one or more chemicals) can be used as a separate step, such as a separate coating and/or repair step.

In some embodiments, the leather preparation process may include treating or restoring the leather with the chemicals described below. In some embodiments, the chemicals described below may be used to treat or restore the leather before or after the drying step. In some embodiments, the chemicals described below may be used to treat or restore the leather before or after the painting step. In some embodiments, the chemicals described below may be used during or as part of the painting step.

In some embodiments, various other steps may be included for specific leather types. In some embodiments, the present disclosure provides methods for making high-quality finished leather (e.g., high-quality black leather and plongé leather). With respect to the production of high-quality finished leather (e.g., high-quality black leather), in some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a dyeing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a drying process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a mechanical stretching process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after a finishing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after a polishing process, or as part of a polishing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after a paint spraying process, or as part of a paint spraying process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before or after a chemical fixing process, or as part of a chemical fixing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a stamping process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a silicone coating step of a coating process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a Uniflex process.

With respect to the production of plongé leather, in some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair the leather before, after, or as part of the dyeing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair the leather before, after, or as part of the drying process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair the leather before, after, or as part of the mechanical stretching process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair the leather before, after, or as part of the mechanical stretching process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair the leather before, after, or as part of the finishing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a first polishing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a color painting process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a second polishing process. In some embodiments, the silk compositions described herein (with or without one or more chemicals) can be used to treat or repair leather before, after, or as part of a Finiflex process.

In some embodiments, the silk compositions that can be used to coat or repair leather and/or leather products according to the processes described herein may include one or more of the silk compositions listed in Table 1.

In one embodiment, the present disclosure provides a method for treating or repairing leather with the silk composition described herein, wherein the method may include the following steps: dyeing the leather; mechanically stretching the leather; trimming the leather; polishing the leather; applying (optionally by spraying) pigments and/or acrylics; chemically fixing the leather, stamping the leather, applying a silicone finish to the leather; and/or providing the leather with a Uniflex treatment; wherein one or more of the foregoing steps includes applying the silk composition to the leather before, during, or after the enumerated steps.

In one embodiment, the present disclosure provides a method for treating or repairing leather with the silk composition described herein, wherein the method may include the following steps: dyeing the leather, drying the leather; mechanically stretching the leather; finishing the leather; performing a first polishing of the leather; applying (optionally by spraying) a colorant and/or acrylic; performing a second polishing of the leather, and/or providing the leather with a Finiflex treatment; wherein one or more of the aforementioned steps includes applying the silk composition to the leather before, during, or after the enumerated steps.

In some embodiments of the methods described herein, the silk compositions described herein can be integrated into the leather processing process (e.g., during, before, or after: pigment + acrylic, pigment + acrylic spraying, colorant spraying, dyeing, fixing spraying, finishing spraying). In some embodiments, the silk compositions described herein can be applied at any part of the larger leatherization process described in Figure 3. In some embodiments of the aforementioned methods, drying can be performed on the leather material that has been sprayed manually or automatically. In some embodiments, a drying step can be provided after and/or before each spraying of the leather material. In some embodiments, the leather material can be dried in an oven. In some embodiments, the drying process may be at a temperature of less than about 70, 71, 72, 73, 74, or 75° C.; or greater than about 70, 71, 72, 73, 74, or 75° C.; or about 70, 71, 72, 73, 74, or 75° C. In some embodiments, each drying step of the leather material may last for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 seconds; or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 seconds; or a period of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 seconds.

In some embodiments of the aforementioned methods, a press can be used during the primary production process by pressing the leather material between a top plate and a bottom plate. In some embodiments, the top plate can be at an operating temperature of less than about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C; or greater than about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C; or about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C. In some embodiments, the pressing step may include pressing the leather material between the first and second plates at the top plate temperature for less than about 1, 2, 3, 4, or 5 seconds; or for more than about 1, 2, 3, 4, or 5 seconds; or for a period of about 1, 2, 3, ⁴ , or 5 seconds. In some embodiments, the pressing step may include pressing the leather material between the first and second plates at the top plate temperature at a pressure of about 75 to about 125 kg/ ^cm2 , or about 90 to about 110 kg/ ^cm2 , or about 100 kg/cm2.

In some embodiments of the foregoing methods, the Finiflex treatment may include compressing the leather material between two heated rotating metal wheels at a temperature of about 75 to about 125° C. or about 93° C. and a pressure of about 5 to about 30 kg/m ² or about 20 kg/m ² for a period of about 1 to about 10 seconds or about 4 seconds.

In some embodiments of the foregoing method, the Uniflex process includes pressing the leather material through two pressing rollers, wherein the top roller is heated to a temperature of about 50°C to about 100°C or about 60°C, and the bottom roller may be unheated, and the two rollers compress the leather material at a pressure of about 10 bar to about 50 bar or about 30 bar for a period of about 1 second to about 10 seconds or about 3 seconds to about 5 seconds.

In some embodiments, the coated leather material prepared by the aforementioned method can be subjected to mechanical quality tests according to one or more of the Veslic method, the Martindale method, the water drop method, the hydration test, and the UV test.

Veslic method - Dry (n=50) and wet (n=10) cycles at f=1.0 Hz, 1 ^cm² abrasion cube applied at 1 kg/ ^cm² . Visual rating of 0-5 (leather and abrasion cube) based on the amount of color removed from the leather and rubbed onto the cube. In some embodiments, the dry cycle may be 0-100; the wet cycle may be 0-30; the frequency may be 0.1-2 Hz; and the pressure may be 0-5 kg/ ^cm² .

Martindale method : A 11 ^cm² circular cut of a leather sample is rubbed against an abrasive in a lissajous pattern (Bowditch curve shape) at a frequency of 0.66-1.0 Hz and 9 kPa for n = 1500 cycles. A visual rating of 0-5 is given based on the amount of color removed from the leather and rubbed onto the cube. In some embodiments, the number of cycles may be 0-5000; the frequency may be 0.1-2 Hz; and the pressure may be 0-50 kPa.

Water Drop Method - 2-4 drops of liquid are run down the length of a vertically oriented leather sample; after 1 minute, if water streaks remain on the surface, the sample is rejected. A visual score of 0-5 is assigned based on the appearance of water streaks on the leather.

Hydration Test - Two circular replicas of the same leather sample are pressed face-to-face with a 300 g weight in a humidity chamber (90% residual humidity; 50°C) for 72 hours. The samples are scored based on how easily they separate from each other and whether any color rubs off. In some embodiments, the weight may be 0-1 kg; the residual humidity may be 70-95%; the temperature may be 40-80°C; and the time may be 24-100 hours.

UV Test - Expose the sample to UV light for 25 hours and observe for color loss. Xe lamp: 42 W/ ^m² , 50°C, λ _incident = 300-400 nm. A visual rating of 0-5 is assigned based on the degree of discoloration of the leather during the test. In some embodiments, the time period may be 20-40 hours; the lamp intensity may be 20-60 W/ ^m² ; the temperature may be 40-80°C; and the λ _incident may be approximately 250-450 nm.

In one embodiment, applying a matte coating composition described herein during the finishing stage (high-quality finishing process) provides a leather product having a matte appearance. In another embodiment, applying a water-soluble dye-fixing coating composition described herein during the finishing stage (high-quality finishing process) provides a leather product dyed with a water-soluble dye. In one embodiment, the leather product is an aniline leather product dyed with a water-soluble aniline leather dye. Polyamide dendrimer for attaching a silk fibroin fragment composition to a cellulose derivative

In some embodiments, a polyamidoamine compound, such as a dendrimer, can be used to adhere a silk fibroin fragment composition or any other coating composition to a cellulose derivative composition and/or coating. In some embodiments, the polyamidoamine compound is Cartaretin F liquid, an aqueous solution of polyamidoamine. In some embodiments, the polyamidoamine compound is cationic. In some embodiments, the cellulose derivative is methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, or microcrystalline cellulose. In some embodiments, the polyamidoamine dendrimer is diluted to a concentration of 0% to 0.1%, 0.1% to 0.2%, 0.2% to 0.3%, 0.3% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1.0%, 1.0% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, or 1.4% to 1.5%. In some embodiments, the dosage of the polyamide amine dendrimer is 0.05% to 0.10%, 0.10% to 0.15%, 0.15% to 0.20%, 0.20% to 0.25%, 0.25% to 0.30%, 0.30% to 0.35%, 0.35% to 0.40%, 0.40% to 0.45%, or 0.45% to 0.50%.

In some embodiments, the silk can be used to paint or repair leather variants that require lighter color treatments. The lighter volume of colorant and pigment used allows the silk to lock the color more effectively.

In some embodiments, the silk can be used in the wet stage of high-quality finished leather processing (e.g., in a small volume mixing drum) to replace another chemical during the colorant mixing stage.

In some embodiments, silk wax (or other silk compositions described herein) can be used to remove defects/pores in raw leather (originating from hair follicles or raw material-related defects) by applying the silk material to the leather at any point along the treatment process. If performed early in the process, this can be used to change the quality grade of the pre-treated leather being selected to produce a higher-quality final product. This effectively provides increased yield (the amount of leather that can be used for a given quality final product). Dyeing Auxiliary

In some embodiments, a dye auxiliary (e.g., Optifix E50 liquid) is used to improve the wet fastness properties of dyed leather. In some embodiments, the dye auxiliary is an aliphatic polyamine. In some embodiments, the aliphatic polyamine is cationic. The dye auxiliary can be applied after the dyeing of the leather or during the intermediate stage (top dyeing). In some embodiments, the dye auxiliary is applied at the wet end stage.

In some embodiments, the dye auxiliary is applied at 30°C, 35°C, 40°C, 45°C, or 50°C. In some embodiments, the dye auxiliary is applied with an acid (e.g., formic acid) and water. Mushroom-based materials

In some embodiments, the substrate includes, but is not limited to, a fungal material, a mushroom-based material, a mycelium-based material, or a fungus-based material and/or the like. All terms, including but not limited to, fungal material, mushroom-based material, mycelium-based material, and fungus-based material, are used interchangeably herein.

As used herein, unless otherwise indicated, the terms "mycological material," "mushroom-based material," "mycelium-based material," "fungus-based material," and "fungal biomass" refer, in certain embodiments, to a mass of fungi that has been cultured, fermented, or grown by any suitable process. In certain embodiments, it is expressly understood that fungal biomass can be produced by any of a variety of methods known in the art, including, but not limited to, surface fermentation methods, submerged fermentation methods, and solid substrate submerged fermentation (SSSF) methods.

In some embodiments, fungal leather-simulating materials are made from inactivated fungal biomass. Furthermore, leather-simulating materials according to the present disclosure can be biodegradable, meaning they biodegrade faster than genuine leather under a given set of conditions. Plant-based Materials

In some embodiments, the substrate includes, but is not limited to, plant-based materials. Some non-limiting examples of plant-based leather sources include pineapple, corn, banana, apple, cactus, green tea, coffee grounds, coconut water, flowers, palm leaves, cork, grapes, kombucha, leaves, paper, cotton, cool stones, bark, Japanese paper, agave, hemp, and hemp plants.

Additionally, the plant-based leather materials disclosed herein can be biodegradable, meaning they biodegrade faster than genuine leather under a given set of conditions. Edible Materials

In some embodiments, the substrate includes, but is not limited to, edible materials or food. In some embodiments, the food is selected from the group consisting of powdered food, dry solid food, oily food, perishable goods, vegetables, fruit, meat, eggs, and seafood. In some embodiments, the perishable goods are selected from the group consisting of vegetables, fruit, meat, eggs, and seafood. In some embodiments, the perishable goods are selected from the group consisting of vegetables and fruit. In some embodiments, the perishable goods are vegetables. In some embodiments, the vegetables are carrots. In some embodiments, the perishable goods are fruits. In some embodiments, the fruits are selected from the group consisting of strawberries, oranges, apples, pears, plums, bananas, grapes, and grapefruits. In some embodiments, the fruits are any berries known in the art. In some embodiments, the fruits are any stone fruits known in the art. In some embodiments, the fruit is any pome known in the art. In some embodiments, the fruit is any citrus known in the art. In some embodiments, the fruit is any melon known in the art. In some embodiments, the fruit is any dried fruit known in the art, such as raisins, prunes, dates, apricots, etc. In some embodiments, the fruit is any stone fruit known in the art. In some embodiments, the perishable commodity is any vegetable known in the art. In some embodiments, the perishable commodity is any seed known in the art.

In some embodiments, the perishable commodity is meat. In some embodiments, the meat is poultry, pork, beef, veal, lamb, bison, ostrich, rabbit, game, fish, eel, shellfish, or seafood. In some embodiments, the poultry is selected from the group consisting of chicken, turkey, duck, goose, and pigeon. Textiles

In some embodiments, the substrate includes, but is not limited to, a textile. In one embodiment, the textile includes a synthetic textile including polyester, Mylar, cotton, nylon, polyester-polyurethane copolymer, rayon, acetate, polyaramid (aromatic polyamide), acrylic, ingeo (polylactide), lurex (polyamide-polyester), olefin (polyethylene-polypropylene), and combinations thereof.

In one embodiment, the textile comprises a natural textile, including alpaca fiber, alpaca down, alpaca wool, camel fiber, camel down, camel wool, cotton, cashmere goat down, and sheep fiber, sheep down, and sheep wool. Filling Agents and Particles

In some embodiments, the coating system includes, but is not limited to, a filler. In some embodiments, the filler is selected from the group consisting of starch-derived fillers, calcium carbonate, calcite, aragonite, hexagonal calcite, amorphous alumina, aluminum silicate, talc, clay, kaolin, sepiolite, alumite, and combinations thereof.

In some embodiments, the coating system includes, but is not limited to, particles. Particles may include polymer particles, mica, silica, mud, and clay. In some embodiments, the substrate comprises clay particles. Throughout this specification, the term "clay" is intended to refer to finely divided earthy materials that become plastic when mixed with water. Clays may be natural, synthetic, or chemically modified. Clays include hydrated aluminum silicates containing small amounts of impurities such as potassium, sodium, magnesium, or iron.

In one embodiment, the clay is a material containing 38.8% to 98.2% SiO2 and 0.3% to 38.0% Al2O3, and further contains one or more metal oxides selected from Fe2O3, CaO, MgO, TiO2, ZrO2, Na2O, and K2O. In some embodiments, the clay has a layered structure comprising hydrous sheets of octahedrally coordinated aluminum, magnesium, or iron, or tetrahedrally coordinated silicon.

In one embodiment, the clay is selected from the group consisting of kaolin, talc, 2:1 phyllosilicate, 1:1 phyllosilicate, bentonite, bentonite, montmorillonite (also known as bentonite), lithium saponite, chromium kaolinite, chlorite, saponite, beidellite, zinc montmorillonite, and mixtures thereof. In one embodiment, the clay is kaolin or bentonite. In some embodiments, the clay is synthetic lithium saponite. In another embodiment, the clay is bentonite.

In some embodiments, the clay has a cation exchange capacity of about 0.7 meq/100 g to about 150 meq/100 g. In some embodiments, the clay has a cation exchange capacity of about 30 meq/100 g to about 100 meq/100 g.

In some embodiments, the coating system optionally comprises composite particles having a cationic charged clay electrostatically complexed with a cationic charged skin conditioning agent as disclosed herein.

Commercially available synthetic lithium saponites include those sold under the trade names Laponite® RD, Laponite® RDS, Laponite® XLG, Laponite® XLS, Laponite® D, Laponite® DF, Laponite® DS, Laponite® S, and Laponite® JS (Southern Clay products, Texas, USA). Commercially available bentonites include those sold under the trade names Gelwhite® GP, Gelwhite® H, Gelwhite® L, Mineral Colloid® BP, Mineral Colloid® MO, Gelwhite® MAS 100(sc), Gelwhite® MAS 101, Gelwhite® MAS 102, Gelwhite® MAS 103, Bentolite® WH, Bentolite® L10, Bentolite® H, Bentolite® L, Permont® SX10A, Permont® SC20 and Permont® HN24 (Southern Clay Products, Texas, USA); Bentone® EW and Bentone® MA (Dow Corning); and Bentonite® USP BL 670 and Bentolite® H4430 (Whitaker, Clarke & Daniels).

In some embodiments, the coating system further comprises a powder component selected from the group consisting of: clay mineral powder, such as talc, mica, sericite, silica, magnesium silicate, synthetic fluorphlogopite, calcium silicate, aluminum silicate, bentonite, montmorillonite; pearl powder, such as aluminum oxide, barium sulfate, dicalcium phosphate, calcium carbonate, titanium oxide, zirconium oxide, zinc oxide, hydroxyapatite, iron oxide, iron titanium oxide, ultramarine blue, Prussian blue, chromium oxide, chromium hydroxide, cobalt oxide, cobalt titanium oxide, titanium oxide coated mica Organic powders, such as polyester, polyethylene, polystyrene, methyl methacrylate resin, cellulose, 12-polyester, 6-polyester, styrene-acrylic acid copolymer, polypropylene, vinyl chloride polymer, tetrafluoroethylene polymer, boron nitride, guanine, tar color dyes, natural color dyes, spherical aluminum oxide, polyacrylates, silicates, sulfates, metal dioxides, carbonates, cellulose, polyalkanes, vinyl acetate, polystyrene, polyamides, acrylic ethers, polysiloxanes, and combinations thereof. Coating system additives

In some embodiments, the coating system further comprises an additive selected from the group consisting of antioxidants, synthetic emulsifiers, solvents, colorants, surfactants (e.g., sophorolipids), astringents, plant extracts, essential oils, cooling agents, humectants, moisturizers, structurants, gelling agents, chelating agents, preservatives, fillers, fragrances, thickeners, humectants, dyes, pigments, shimmering agents, and combinations thereof. Chemicals for use with leather and leather products coated with fibroin-based protein fragments

In certain embodiments, the chemical agent can be used to pre-treat, treat, and/or post-treat the leather or leather products described herein. In certain embodiments, the silk and/or SPF solution (e.g., SFS) or composition described herein can include one or more of the chemical agents described herein. In certain embodiments, the silk and/or SPF solution or composition described herein can replace one or more of the chemical agents described herein. In certain embodiments, the chemical agent can be selected from the group consisting of silicones, caseins, acidic agents, dyes, pigments, traditional finishes, and technical finishes. In certain embodiments, the chemical agent can include one or more of the reagents listed in Table 2. In some embodiments, the chemical agent can be selected from the group consisting of: water-based paint, wax, oil, binder (protein or other), filler, hand modifier, leveling agent, solvent paint, water-based paint, penetrant, acrylic resin, butadiene resin, dense resin, hybrid resin, impregnating resin, rheology modifier, solvent matting agent, solvent urethane, water-based matting agent, water-based topcoat, chromium, acid dye, alkaline dye, dye (chromium or other), colorant, and combinations thereof.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a moisturizer. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a moisturizer. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a moisturizer. In one embodiment, the wetting agent improves one or more coating properties. Suitable wetting agents are known to those skilled in the art. Illustrative, non-limiting examples of wetting agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a cleaning agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a cleaning agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a cleaning agent. In one embodiment, the cleaning agent improves one or more coating properties. Suitable cleaning agents are known to those skilled in the art. Illustrative, non-limiting examples of cleaning agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a chelating or dispersing agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a chelating or dispersing agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a chelating or dispersing agent. Suitable chelating or dispersing agents are known to those skilled in the art. Illustrative, non-limiting examples of chelating or dispersing agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with an enzyme. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with an enzyme. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with an enzyme. Suitable enzymes are known to those skilled in the art. Illustrative, non-limiting examples of enzymes from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a bleaching agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a bleaching agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with a bleaching agent. Suitable bleaching agents are known to those skilled in the art. Illustrative, non-limiting examples of bleaching agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a defoaming agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a defoaming agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is pretreated with a defoaming agent. Suitable defoaming agents are known to those skilled in the art. Illustrative, non-limiting examples of defoamers from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with an anti-wrinkle agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with an anti-wrinkle agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product has been pretreated with an anti-wrinkle agent. Suitable anti-wrinkle agents are known to those skilled in the art. Illustrative, non-limiting examples of anti-wrinkle agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye dispersant. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye dispersant. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye dispersant. Suitable dye dispersants are known to those skilled in the art. Illustrative, non-limiting examples of dye dispersants from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-leveling agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-leveling agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-leveling agent. Suitable dye-leveling agents are known to those skilled in the art. Illustrative, non-limiting examples of dye leveling agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye fixing agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye fixing agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye fixing agent. Suitable dye fixing agents are known to those skilled in the art. Illustrative, non-limiting examples of dye fixing agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-specific resin. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-specific resin. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye-specific resin. Suitable dye-specific resins are known to those skilled in the art. Illustrative, non-limiting examples of dye-specific resins from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye anti-reduction agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye anti-reduction agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a dye anti-reduction agent. Suitable dye anti-reduction agents are known to those skilled in the art. Illustrative, non-limiting examples of dye anti-reduction agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system anti-migration agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system anti-migration agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system anti-migration agent. Suitable pigment dye system anti-migration agents are known to those skilled in the art. Illustrative, non-limiting examples of pigment dye system anti-migration agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system adhesive. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system adhesive. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system adhesive. Suitable pigment dye system binders are known to those skilled in the art. Illustrative, non-limiting examples of pigment dye system binders from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a combination of a pigment system binder and an anti-migration agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a combination of a pigment system binder and an anti-migration agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a pigment system binder and an anti-migration agent combination. Suitable pigment system binder and anti-migration agent combinations are known to those skilled in the art. Illustrative, non-limiting examples of pigment system binder and anti-migration agent combinations from representative supplier Lamberti SPA are provided in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a colorant. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a colorant. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is treated with a colorant. Suitable colorants are known to those skilled in the art. Illustrative, non-limiting examples of rendering agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally treated with a wrinkle-free treatment. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally treated with a wrinkle-free treatment. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally treated with a wrinkle-free treatment. Suitable wrinkle-reducing agents are known to those skilled in the art. Illustrative, non-limiting examples of wrinkle-reducing agents from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a softening agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a softening agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a softening agent. Suitable softeners are known to those skilled in the art. Illustrative, non-limiting examples of softeners from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a feel modifier. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a feel modifier. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been traditionally coated with a feel modifier. Suitable feel modifiers are known to those skilled in the art. Illustrative, non-limiting examples of feel modifiers from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been conventionally coated with an aqueous polyurethane (PU) dispersion. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been conventionally coated with an aqueous polyurethane (PU) dispersion. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product has been conventionally coated with an aqueous polyurethane (PU) dispersion. Suitable aqueous polyurethane dispersions for conventional painting are known to those skilled in the art. Illustrative, non-limiting examples of aqueous polyurethane dispersions for conventional painting from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is conventionally coated with a finishing resin. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is conventionally coated with a finishing resin. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is conventionally coated with a finishing resin. Suitable finishing resins are known to those skilled in the art. Illustrative, non-limiting examples of finishing resins from representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an aqueous polyurethane dispersion. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an aqueous polyurethane dispersion. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an aqueous polyurethane dispersion. Suitable aqueous polyurethane dispersions for technical coatings are known to those skilled in the art. Illustrative, non-limiting examples of aqueous polyurethane dispersions for technical coatings from the representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an oil- or water-repellent agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an oil- or water-repellent agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with an oil- or water-repellent agent. Suitable oil- or water-repellents for technical paints are known to those skilled in the art. Illustrative, non-limiting examples of oil- or water-repellents for technical paints from the representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a flame retardant. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a flame retardant. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a flame retardant. Suitable flame retardants for technical coatings are known to those skilled in the art. Illustrative, non-limiting examples of flame retardants for technical coatings from the representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products processed with a composition comprising a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a crosslinking agent. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a crosslinking agent. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a crosslinking agent. Suitable crosslinking agents for technical coatings are known to those skilled in the art. Illustrative, non-limiting examples of crosslinking agents for technical coatings from the representative supplier Lamberti SPA are given in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a thickener for technical coatings. In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a seryl protein or fragment thereof having an average weight-average molecular weight in the range of about 5 kDa to about 144 kDa, wherein the leather or leather product is technically coated with a thickener for technical coatings. In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a sirtuin protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is technically finished with a thickener for technical finishes. Thickeners suitable for technical finishes are known to those skilled in the art. Exemplary, non-limiting examples of thickeners for technical finishes from representative supplier Lamberti SPA are provided in the table below.

In one embodiment, the present disclosure provides leather or leather products treated with a composition comprising a silk-based protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is coated with one or more of Silky Top 7425 NF, Uniseal 9049, Unithane 351 NF, and Unithane 2132 NF (Union Specialties, Inc.). In one embodiment, the present disclosure provides leather or leather products having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is coated with one or more of Silky Top 7425 NF, Uniseal 9049, Unithane 351 NF, and Unithane 2132 NF (Union Specialties, Inc.). In one embodiment, the present disclosure provides leather or leather products comprising a defect repair filler, wherein the filler comprises a silk-based protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the leather or leather product is coated with one or more of Silky Top 7425 NF, Uniseal 9049, Unithane 351 NF, and Unithane 2132 NF (Union Specialties, Inc.). Other suitable Union Specialties products (such as coatings, additives, and/or oils and waxes) are known to those skilled in the art. Illustrative, non-limiting examples of Union Specialties products are given in the following table:

In any of the aforementioned leather or leather product embodiments, the processing composition comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 5 kDa to about 144 kDa. In any of the aforementioned leather or leather product embodiments, the processing composition comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 6 kDa to about 17 kDa. In any of the aforementioned leather or leather product embodiments, the processing composition comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 17 kDa to about 39 kDa. In any of the aforementioned leather or leather product embodiments, the processing composition comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 39 kDa to about 80 kDa.

In any of the aforementioned leather or leather product embodiments, the coating comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 5 kDa to about 144 kDa. In any of the aforementioned leather or leather product embodiments, the coating comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 6 kDa to about 17 kDa. In any of the aforementioned leather or leather product embodiments, the coating comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 17 kDa to about 39 kDa. In any of the aforementioned leather or leather product embodiments, the coating comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 39 kDa to about 80 kDa.

In any of the aforementioned leather or leather product embodiments, the defect repair filler comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 5 kDa to about 144 kDa. In any of the aforementioned leather or leather product embodiments, the defect repair filler comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 6 kDa to about 17 kDa. In any of the aforementioned leather or leather product embodiments, the defect repair filler comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 17 kDa to about 39 kDa. In any of the aforementioned leather or leather product embodiments, the defect repair filler comprises a seryl protein or fragment thereof having an average weight average molecular weight in the range of about 39 kDa to about 80 kDa.

In any of the aforementioned leather or leather product embodiments, the processing composition comprises low molecular weight silk of silylated proteins or fragments thereof. In any of the aforementioned leather or leather product embodiments, the processing composition comprises medium molecular weight silk. In any of the aforementioned leather or leather product embodiments, the processing composition comprises heavy molecular weight silk. In any of the aforementioned leather or leather product embodiments, the processing composition comprises silylated proteins or fragments thereof, wherein the silylated proteins or fragments thereof comprise one or more of low, medium, and high molecular weight silk.

In any of the aforementioned leather or leather product embodiments, the coating comprises low molecular weight silks of silylated proteins or fragments thereof. In any of the aforementioned leather or leather product embodiments, the coating comprises medium molecular weight silks. In any of the aforementioned leather or leather product embodiments, the coating comprises heavy molecular weight silks. In any of the aforementioned leather or leather product embodiments, the coating comprises silylated proteins or fragments thereof, wherein the silylated proteins or fragments thereof comprise one or more of low, medium, and high molecular weight silks.

In any of the aforementioned leather or leather product embodiments, the defect-repairing filler comprises low molecular weight silk of a silylated protein or a fragment thereof. In any of the aforementioned leather or leather product embodiments, the defect-repairing filler comprises medium molecular weight silk. In any of the aforementioned leather or leather product embodiments, the defect-repairing filler comprises heavy molecular weight silk. In any of the aforementioned leather or leather product embodiments, the defect-repairing filler comprises silylated protein or a fragment thereof, wherein the silylated protein or fragment thereof comprises one or more of low, medium, and high molecular weight silk.

In any of the aforementioned leather or leather product embodiments, the fibroin or protein fragment thereof has an average weight average molecular weight range selected from the group consisting of: about 5 to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the fibroin or protein fragment thereof has a polydispersity between about 1.5 and about 3.0, and optionally wherein the protein or protein fragment does not spontaneously or gradually gel and does not significantly change in color or turbidity when in solution for at least 10 days prior to processing, coating, and/or repairing the leather or leather product. Process for producing fibroin-based protein fragments and solutions thereof

As used herein, the term "silk fibroin" includes both silk fibroin and insect or spider silk proteins. In one embodiment, the silk fibroin is obtained from Bombyx mori. In one embodiment, the spider silk protein is selected from the group consisting of: sheath silk (grape silk), egg sac silk (cylindrical silk), egg shell silk (tubular silk), non-adhesive drag silk (potential silk), attachment thread silk (pyriform silk), adhesive core silk (flagellate silk), and adhesive outer silk (aggregate silk).

Silk-based proteins or fragments thereof, silk solutions or mixtures (e.g., SPF or SFS solutions or mixtures), and their analogs can be prepared according to the methods described in U.S. Patent Nos. 9,187,538, 9,522,107, 9,522,108, 9,511,012, 9,517,191, 9,545,369, and 10,166,177, as well as U.S. Patent Publication Nos. 2016/0222579 and 2016/0281294, and International Patent Publication Nos. WO 2016/090055 and WO 2017/011679, the entire contents of which are incorporated herein by reference. In some embodiments, the silk-based protein or its fragments may be provided as a silk composition, which may be an aqueous solution or mixture of silk, silk gel, and/or silk wax as described herein. Methods for using silk fibroin or silk fibroin fragments in coating applications are known and described, for example, in U.S. Patent Nos. 10,287,728 and 10,301,768.

The following are non-limiting examples of suitable ranges of various parameters in and for preparing the silk solutions and/or compositions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of such parameter ranges.

In one embodiment, the percentage of silk in the solution or composition is undetectable to 30%. In one embodiment, the percentage of silk in the solution or composition is undetectable to 5%. In one embodiment, the percentage of silk in the solution or composition is 1%. In one embodiment, the percentage of silk in the solution or composition is 2%. In one embodiment, the percentage of silk in the solution or composition is 3%. In one embodiment, the percentage of silk in the solution or composition is 4%. In one embodiment, the percentage of silk in the solution or composition is 5%. In one embodiment, the percentage of silk in the solution or composition is 10%. In one embodiment, the percentage of silk in the solution or composition is 30%.

In one embodiment, the solution or composition of the present disclosure comprises pure fibrous protein fragments having an average weight-average molecular weight in the range of 6 kDa to 17 kDa. In one embodiment, the solution or composition of the present disclosure comprises pure fibrous protein fragments having an average weight-average molecular weight in the range of 17 kDa to 39 kDa. In one embodiment, the solution or composition of the present disclosure comprises pure fibrous protein fragments having an average weight-average molecular weight in the range of 39 kDa to 80 kDa.

In one embodiment, the compositions of the present disclosure include silk protein fragments having an average weight-average molecular weight in the range of 6 kDa to 17 kDa. In one embodiment, the compositions of the present disclosure include silk protein fragments having an average weight-average molecular weight in the range of 17 kDa to 39 kDa. In one embodiment, the compositions of the present disclosure include silk protein fragments having an average weight-average molecular weight in the range of 39 kDa to 80 kDa.

In one embodiment, the compositions of the present disclosure include silk protein fragments having an average weight average molecular weight of about 1 kDa to about 350 kDa, or about 1 kDa to about 300 kDa, or about 1 kDa to about 250 kDa, or about 1 kDa to about 200 kDa, or about 1 kDa to about 150 kDa, or about 1 kDa to about 100 kDa, or about 1 kDa to about 50 kDa, or about 1 kDa to about 25 kDa.

In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight in the range of 1 kDa to 6 kDa. In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight in the range of 6 kDa to 16 kDa. In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight in the range of 16 kDa to 38 kDa. In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight in the range of 38 kDa to 80 kDa. In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight ranging from 80 kDa to 150 kDa.

In one embodiment, the silk fibroin-based protein fragments incorporated into the silk compositions described herein have an average weight average molecular weight ranging from 1 kDa to 250 kDa.

In some embodiments, the filament compositions provided herein can be applied to the product to be processed in a mixture or in a stepwise process. For example, a filament composition comprising a low molecular weight filament and a medium molecular weight filament can be applied to the product to be processed. Alternatively, a low molecular weight filament composition can be applied to the product to be processed, as provided by the process described herein, and then a medium or high molecular weight filament can be applied to the product. The low, medium, and high molecular weight filament compositions can be added in any order or in any combination (e.g., low/medium, low/high, medium/high, low/medium/high).

In some embodiments, the silk composition provided herein can be applied to the article to be coated in the form of a mixture or applied in a step-by-step process to form a coating on the article. For example, a silk composition comprising low molecular weight silk and medium molecular weight silk can be applied to the article to be coated. Alternatively, a low molecular weight silk composition can be applied to the article to be coated, as provided by the process described herein, and then medium or high molecular weight silk can be applied to the article. Low, medium and high molecular weight silk compositions can be added in any order or in any combination (e.g., low/medium, low/high, medium/high, low/medium/high).

In some embodiments, the filament compositions provided herein can be applied to the article to be repaired as a mixture or in a stepwise process to form a filler in or on the article. For example, a filament composition comprising low molecular weight filaments and medium molecular weight filaments can be applied to the article to be repaired. Alternatively, a low molecular weight filament composition can be applied to the article to be repaired, as provided by the process described herein, and then medium or high molecular weight filaments can be applied to the article. The low, medium, and high molecular weight filament compositions can be added in any order or in any combination (e.g., low/medium, low/high, medium/high, low/medium/high).

In some embodiments, where multiple layers of the filament composition are applied to the article to be coated, it may have at least one layer, or 1 to 1 million layers, or 1 to 100,000 layers, or 1 to 10,000 layers, or 1 to 1,000 layers of such a filament composition, wherein the layers may have the same or different thicknesses. For example, in some embodiments, a layer can have a thickness of about 1 nm to about 1 mm, or about 1 nm to about 1 μm, or about 1 nm to about 500 nm, or about 1 nm to about 400 nm, or about 1 nm to about 300 nm, or about 1 nm to about 200 nm, or about 1 nm to about 100 nm, or about 1 nm to about 75 nm, or about 1 nm to about 50 nm, or about 1 nm to about 25 nm, or about 1 nm to about 20 nm, or about 1 nm to about 15 nm, or about 1 nm to about 10 nm, or about 1 nm to about 5 nm.

In one embodiment, the composition of the present disclosure having pure fibrous protein-based protein fragments has a polydispersity in the range of about 1 to about 5.0. In one embodiment, the composition of the present disclosure having pure fibrous protein-based protein fragments has a polydispersity in the range of about 1.5 to about 3.0. In one embodiment, the composition of the present disclosure having pure fibrous protein-based protein fragments has a polydispersity in the range of about 1 to about 1.5. In one embodiment, the composition of the present disclosure having pure fibrous protein-based protein fragments has a polydispersity in the range of about 1.5 to about 2.0. In one embodiment, the composition of the present disclosure having pure fibrous protein-based protein fragments has a polydispersity in the range of about 2.0 to about 2.5. In one embodiment, the composition of the present disclosure having pure fibrous protein fragments has a polydispersity ranging from about 2.0 to about 3.0. In one embodiment, the composition of the present disclosure having pure fibrous protein fragments has a polydispersity ranging from about 2.5 to about 3.0.

In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 1 to about 5.0. In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 1.5 to about 3.0. In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 1 to about 1.5. In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 1.5 to about 2.0. In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 2.0 to about 2.5. In one embodiment, the composition comprising a silk protein fragment of the present disclosure has a polydispersity in the range of about 2.0 to about 3.0. In one embodiment, the disclosed compositions having silk protein fragments have a polydispersity ranging from about 2.5 to about 3.0.

In some embodiments, the polydispersity of the low molecular weight filament protein fragments can be from about 1 to about 5.0, or from about 1.5 to about 3.0, or from about 1 to about 1.5, or from about 1.5 to about 2.0, or from about 2.0 to about 2.5, or from about 2.5 to about 3.0.

In some embodiments, the polydispersity of the medium molecular weight filament protein fragments can be from about 1 to about 5.0, or from about 1.5 to about 3.0, or from about 1 to about 1.5, or from about 1.5 to about 2.0, or from about 2.0 to about 2.5, or from about 2.5 to about 3.0.

In some embodiments, the polydispersity of the high molecular weight silk protein fragments can be from about 1 to about 5.0, or from about 1.5 to about 3.0, or from about 1 to about 1.5, or from about 1.5 to about 2.0, or from about 2.0 to about 2.5, or from about 2.5 to about 3.0.

In some embodiments, in the compositions described herein having a combination of low-, medium-, and/or high-molecular-weight silk protein fragments, such low-, medium-, and/or high-molecular-weight silk proteins may have the same or different polydispersities. Bio-based Polyurethanes

In some embodiments, the coating system includes, but is not limited to, a bio-based polyurethane. In some embodiments, the bio-based polyurethane is biodegradable. Biodegradable polyurethanes can be obtained using a biodegradable soft segment and an isomannitol hard segment. In the biodegradable soft segment, polyurethanes such as polyurethanes containing poly(ε-caprolactone) (PCL), as well as poly(ethylene oxide) (PEO) and poly(l-lactide) PLA have been obtained. In the biodegradable hard segment, diisocyanates and chain extenders can be designed from a variety of biorelevant molecules. Some non-limiting examples of bio-based polyurethanes are further described at sciencedirect.com/topics/engineering/biodegradable- polyurethane and ncbi.nlm.nih.gov/pmc/articles/ PMC4108296/. Compositions and Processes Including Fiber Protein-Based Processing Compositions, Coatings, or Fillers

In one embodiment, the present disclosure may include leather or leather products that can be processed, coated, or repaired with an SPF mixture solution (i.e., a silk fibroin solution (SFS)) and/or composition as described herein to produce a processed, coated, or repaired product. In one embodiment, the processed, coated, or repaired product described herein can be treated with additional chemicals that enhance the properties of the coated product. In one embodiment, the SFS can enhance the properties of the coated or repaired product, or the SFS can include one or more chemicals that enhance the properties of the coated or repaired product.

In some embodiments, chemical coatings may be applied to leather or leather products before or after they have been processed, coated, or repaired with SFS. In one embodiment, chemical coatings may involve applying chemicals and/or SFS to leather or leather products to alter the properties of the original leather or leather product and to impart properties not originally present in the leather or leather product. In the case of chemical coatings, the leather or leather product treated with such chemical coatings may act as a surface treatment and/or such treatments may alter the elemental analysis of the base polymer of the treated leather or leather product.

In one embodiment, one type of chemical finishing may involve applying certain fibroin-based solutions to leather or leather products. For example, the SFS may be applied to the leather or leather product after it has been dyed, but there are also situations where the SFS may need to be applied during processing, during dyeing, or after a garment has been assembled from the selected leather or leather product. In some embodiments, after application, the SFS may be dried using heat. In some embodiments, the SFS may then be fixed to the surface of the leather or leather product in a process step known as curing.

In some embodiments, the SFS can be provided in a concentrated form suspended in water. In some embodiments, the SFS can have a concentration of less than about 50% by weight (% w/w or % w/v), or less than about 45%, or less than about 40%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.1%, or less than about 0.01%, or less than about 0.001%, or less than about 0.0001%, or less than about 0.00001%. In some embodiments, the SFS may have a concentration of greater than about 50% by weight (% w/w or % w/v), or greater than about 45%, or greater than about 40%, or greater than about 35%, or greater than about 30%, or greater than about 25%, or greater than about 20%, or greater than about 15%, or greater than about 10%, or greater than about 5%, or greater than about 4%, or greater than about 3%, or greater than about 2%, or greater than about 1%, or greater than about 0.1%, or greater than about 0.01%, or greater than about 0.001%, or greater than about 0.0001%, or greater than about 0.00001%.

In some embodiments, the amount of a fibrous protein solution (SFS) is determined by the solution concentration and the material's wet absorption rate. The fibrous protein solution may include fibroin or fragments thereof that can be fixed or otherwise attached to the coated leather or leather product. The wet absorption rate can be represented by the following formula: The total amount of SFS added to leather or leather products can be expressed as follows:

In one embodiment, the silk-based protein film is naturally derived, renewable, and biodegradable. Without wishing to be bound by any particular theory, it is believed that the expanded application of silk-based biomaterials can potentially replace synthetic chemicals and promote the sustainability and safety of commercial products. In most cases, silk crystallizes and forms a rigid structure with a high Young's modulus and low elongation at break. This is mainly attributed to interchain hydrogen bonds and hydrophobic interactions. This compact and crystalline structure can be broken down and reconstituted into an amorphous structure, which is a soft and flexible structure of the silk-based protein.

In one example, a brittle/rigid film is converted into a flexible film by adding a plasticizer such as glycerol. Glycerol disrupts non-covalent interchain bonding; thus, it creates spaces between protein chains and reduces "chain friction." However, this technique has limitations. Adding too much glycerol can over-plasticize the protein and make it difficult to form a film. Adding a moderate amount of glycerol only increases stretchability from 4% to 40%, which is still lower than commercial stretchable leather finish resins with elongations exceeding 500%.

In one embodiment, fibroin interactions can be disrupted by adding salt. Salt has a strong charge and is highly soluble in water, allowing it to interact strongly with protein segments. Specifically, anions interact with positively charged NH3+, while cations interact with ^-COO- . This strong electrostatic attraction prevents the protein from forming β-sheets, which contribute to its brittle crystalline structure.

In some embodiments, the incorporation of salts can improve the flexibility of silk films.

Regarding methods of applying SFS to leather or leather products, more generally, SFS can be applied to leather or leather products via a pad or roll application process, a soak and remove process, and/or a topical application process. Furthermore, methods of wire application (i.e., SFS application or coating) can include bath application, kiss roll application, spray application, and/or double-sided roll application. In some embodiments, the coating process (e.g., bath application, kiss roll application, spray application, double-sided roll application, roll application, soak and remove application, and/or topical application), drying process, and curing process can be varied as described herein to alter one or more selected leather or leather product properties of the resulting coated leather or leather product, including such properties.

In one embodiment, the drying and/or curing temperature of the disclosed process may be less than about 70°C, or less than about 75°C, or less than about 80°C, or less than about 85°C, or less than about 90°C, or less than about 95°C, or less than about 100°C, or less than about 110°C, or less than about 120°C, or less than about 130°C, or less than about 140°C, or less than about 150°C, or less than about 160°C, or less than about 170°C, or less than about 180°C, or less than about 190°C, or less than about 200°C, or less than about 210°C, or less than about 220°C, or less than about 230°C.

In one embodiment, the drying and/or curing temperature of the disclosed process may be greater than about 70°C, or greater than about 75°C, or greater than about 80°C, or greater than about 85°C, or greater than about 90°C, or greater than about 95°C, or greater than about 100°C, or greater than about 110°C, or greater than about 120°C, or greater than about 130°C, or greater than about 140°C, or greater than about 150°C, or greater than about 160°C, or greater than about 170°C, or greater than about 180°C, or greater than about 190°C, or greater than about 200°C, or greater than about 210°C, or greater than about 220°C, or greater than about 230°C.

In one embodiment, the drying time of the disclosed process may be less than about 10 seconds, or less than about 20 seconds, or less than about 30 seconds, or less than about 40 seconds, or less than about 50 seconds, or less than about 60 seconds, or less than about 2 minutes, or less than about 3 minutes, or less than about 4 minutes, or less than about 5 minutes, or less than about 6 minutes, or less than about 7 minutes, or less than about 8 minutes, or less than about 9 minutes, or less than about 10 minutes, or less than about 20 minutes, or less than about 30 minutes, or less than about 40 minutes, or less than about 50 minutes, or less than about 60 minutes.

In one embodiment, the drying time of the process of the present disclosure may be greater than about 10 seconds, or greater than about 20 seconds, or greater than about 30 seconds, or greater than about 40 seconds, or greater than about 50 seconds, or greater than about 60 seconds, or greater than about 2 minutes, or greater than about 3 minutes, or greater than about 4 minutes, or greater than about 5 minutes, or greater than about 6 minutes, or greater than about 7 minutes, or greater than about 8 minutes, or greater than about 9 minutes, or greater than about 10 minutes, or greater than about 20 minutes, or greater than about 30 minutes, or greater than about 40 minutes, or greater than about 50 minutes, or greater than about 60 minutes.

In one embodiment, the curing time of the disclosed process may be less than about 1 second to less than about 60 minutes.

In one embodiment, the curing time of the disclosed process may be greater than about 1 second to greater than about 60 minutes.

In some embodiments, a material processed or coated with filamentous protein can be heat resistant to a selected temperature, wherein the selected temperature is selected for drying, curing and/or heat setting a dye that can be applied to the material (e.g., coated leather or leather products). As used herein, "heat resistance" can refer to a property of a fibrous protein coating deposited on a material, wherein the fibrous protein coating and/or fibrous protein does not exhibit a significant change in the performance of the fibrous protein coating (i.e., "substantially changed") compared to a control material having a comparable fibrous protein coating that has not been subjected to a selected temperature for drying, curing, washing cycles, and/or heat setting purposes. In some embodiments, the selected temperature is the glass transition temperature (Tg) of the material to which the fibrous protein coating is applied. In some embodiments, the selected temperature is greater than about 65°C, or greater than about 70°C, or greater than about 80°C, or greater than about 90°C, or greater than about 100°C, or greater than about 110°C, or greater than about 120°C, or greater than about 130°C, or greater than about 140°C, or greater than about 150°C, or greater than about 160°C, or greater than about 170°C, or greater than about 180°C, or greater than about 190°C, or greater than about 200°C, or greater than about 210°C, or greater than about 220°C. In some embodiments, the selected temperature is less than about 65°C, or less than about 70°C, or less than about 80°C, or less than about 90°C, or less than about 100°C, or less than about 110°C, or less than about 120°C, or less than about 130°C, or less than about 140°C, or less than about 150°C, or less than about 160°C, or less than about 170°C, or less than about 180°C, or less than about 190°C, or less than about 200°C, or less than about 210°C, or less than about 220°C.

In some embodiments, the SFS-processed, coated, or repaired article may be heat set to set one or more dyes that may be applied to the SFS-coated article, thereby permanently setting the one or more dyes on the SFS-coated or repaired article. In some embodiments, the SFS-processed, coated, or repaired article may be heat-set resistant, wherein the SFS coating on the SFS-coated article may withstand heat-setting temperatures greater than about 100°C, or greater than about 110°C, or greater than about 120°C, or greater than about 130°C, or greater than about 140°C, or greater than about 150°C, or greater than about 160°C, or greater than about 170°C, or greater than about 180°C, or greater than about 190°C, or greater than about 200°C, or greater than about 210°C, or greater than about 220°C. In some embodiments, the selected temperature is less than about 100°C, or less than about 110°C, or less than about 120°C, or less than about 130°C, or less than about 140°C, or less than about 150°C, or less than about 160°C, or less than about 170°C, or less than about 180°C, or less than about 190°C, or less than about 200°C, or less than about 210°C, or less than about 220°C.

In one embodiment, after the fibrous protein coated or repaired material is subjected to heating and/or curing as described herein, the material processed, coated, or repaired with the fibrous protein coating or filling composition as described herein can partially dissolve or otherwise partially incorporate into a portion of the material. Without being limited by any theory, the fibrous protein coating can partially dissolve or otherwise partially incorporate into a portion of the material when the fibrous protein coated or repaired material is heated to a temperature greater than about the glass transition temperature (Tg) of the processed, coated, or repaired material.

In some embodiments, the material processed, coated, or repaired with a silk fibroin coating as described herein can be sterile or can be sterilized to provide a sterilized silk fibroin-coated material. Alternatively or in addition, the methods described herein can include a sterile SFS prepared from sterile silk fibroin.

In some embodiments, SFS can be used in SFS processing compositions, coatings, or repair compositions, wherein such compositions or coatings include one or more chemicals (e.g., silicones). SFS can be measured by weight (% w/w or % w/v), or a concentration of less than about 50%, or less than about 45%, or less than about 40%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 9%, or less than about 8%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.9%, or less than about 0.8%, or less than about 0.7%, or less than about 0.6%, or less than about 0.5%, or less than about 0.4%, or less than about 0.3%, or less than about 0.2%, or less than about 0.1%, or less than about 0.01%, or less than about 0.001% by volume (v/v). In some embodiments, SFS may be provided in such SFS coatings at a concentration of greater than about 25% by weight (% w/w or % w/v), or greater than about 20%, or greater than about 15%, or greater than about 10%, or greater than about 9%, or greater than about 8%, or greater than about 7%, or greater than about 6%, or greater than about 5%, or greater than about 4%, or greater than about 3%, or greater than about 2%, or greater than about 1%, or greater than about 0.9%, or greater than about 0.8%, or greater than about 0.7%, or greater than about 0.6%, or greater than about 0.5%, or greater than about 0.4%, or greater than about 0.3%, or greater than about 0.2%, or greater than about 0.1%, or greater than about 0.01%, or greater than about 0.001%.

In some embodiments, the chemical fabric softener may include silicone as described herein.

In some embodiments, the chemicals may include the following chemicals, which are supplied by CHT Bezema and are associated with the properties of certain selected leathers or leather products and which may be used to enhance the adhesion of the SFS to the painted or repaired surface and/or the SFS may be used to enhance the properties of the following chemicals: ALPAPRINT CLEAR Silicone printing and coating Component B is mentioned in the technical brochure Dry feel Good rubbing fastness Good wash fastness ALPAPRINT ELASTIC ADD Silicone printing and coating Component B is mentioned in the technical brochure Good rubbing fastness Good wash fastness Suitable for yardage printing and dyeing ALPAPRINT WHITE Silicone printing and coating Component B is mentioned in the technical brochure Dry feel Good rubbing fastness Good wash fastness ALPATEC 30142 A Textile coating painting Silicone printing and coating Component B is mentioned in the technical brochure Suitable for narrow band coating Good rubbing fastness Good wash fastness ALPATEC 30143 A Silicone printing and coating Component B is mentioned in the technical brochure Good rubbing fastness Good wash fastness Suitable for yardage printing and dyeing ALPATEC 30191 A Silicone printing and coating Component B is mentioned in the technical brochure Suitable for narrow band coating High transparency painting ALPATEC 30203 A Silicone printing and coating Component B is mentioned in the technical brochure Suitable for narrow band coating High transparency painting ALPATEC 3040 LSR KOMP.A Functional coatings, silicone printing and coating Component B is mentioned in the technical brochure High wear resistance High transparency painting ALPATEC 3060 LSR KOMP.A Functional coatings, silicone printing and coating Component B is mentioned in the technical brochure High wear resistance High transparency painting ALPATEC 530 Silicone printing and coating Suitable for narrow band coating High transparency painting One-component system ALPATEC 540 Silicone printing and coating Suitable for narrow band coating High transparency painting One-component system ALPATEC 545 Silicone printing and coating Suitable for narrow band coating High transparency painting One-component system ALPATEC 550 Silicone printing and coating Suitable for narrow band coating High transparency painting One-component system ALPATEC 730 Silicone printing and coating Suitable for narrow band coating Good wash fastness High wear resistance High transparency ALPATEC 740 Silicone printing and coating Suitable for narrow band coating Good wash fastness High wear resistance High transparency ALPATEC 745 Silicone printing and coating Suitable for narrow band coating Good wash fastness High wear resistance High transparency ALPATEC 750 Silicone printing and coating Suitable for narrow band coating Good wash fastness High wear resistance High transparency ALPATEC BANDAGE A Silicone printing and coating Component B is mentioned in the technical brochure Suitable for narrow band coating painting Two-component system APYROL BASE2 E flame retardants liquid Soft feel Applicable to BS 5852/1+2 Suitable for paste coatings APYROL FCR-2 Water/oil repellency cations Efficient Water-based liquid APYROL FFD E flame retardants liquid Suitable for polyester Applicable to polyamide flame retardant filler APYROL FR CONC E Flame retardants, functional coatings liquid Suitable for polyester Applicable to polyamide flame retardant filler APYROL GBO-E Flame retardants, functional coatings Suitable for polyester Light-blocking paint Applicable to DIN 4102/B1 Contains halogens APYROL LV 21 Flame retardants, functional coatings Applicable to DIN 4102/B1 Suitable for paste coatings For back coating of blackout vertical blinds and roller blinds Contains halogens APYROL PP 31 flame retardants liquid Does not contain antimony flame retardant filler Applicable to BS 5852/1+2 APYROL PP 46 flame retardants powder Does not contain antimony flame retardant filler Suitable for paste coatings APYROL PREM E flame retardants Soft feel Applicable to BS 5852/1+2 Contains halogens semi-permanent APYROL PREM2 E flame retardants Soft feel Applicable to BS 5852/1+2 Contains halogens semi-permanent COLORDUR 005 WHITE Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 105 LEMON Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 115 GOLDEN YELLOW Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 185 ORANGE Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 215 RED Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 225 DARK RED Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 285 VIOLET Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 305 BLUE Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 355 MARINE Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 405 GREEN Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 465 OLIVE GREEN Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR 705 BLACK Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension Colorur AM Additive Velvet adhesive, silicone printing and coating Silicone-based Migration Prevention Dye pigment suspension COLORDUR FL 1015 YELLOW Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR FL 1815 ORANGE Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR FL 2415 PINK Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension COLORDUR FL 4015 GREEN Planting adhesives, functional coatings, polysilicone printing and coating Silicone-based Dye pigment suspension ECOPERL 1 Water/oil repellency Washable Sprayable Based on special functionalized polymers/waxes cations ECOPERL ACTIVE Water/oil repellency Washable Based on special functionalized polymers/waxes cations Efficient LAMETHAN 1 ET 25 BR 160 Functional coating, lamination Washable transparent 25 µm strength Polyester urethane based membrane LAMETHAN ADH-1 Functional coating, lamination breathable Suitable for dry lamination Good washing stability at 40°C Stable foam adhesive LAMETHAN ADH-L Functional coating, lamination Washable transparent Suitable for paste coatings Suitable for wet lamination LAMETHAN ALF-K Functional coating, lamination Adhesive additives for bonding Suitable for dry lamination Stable foam adhesive Suitable for stable foam coatings LAMETHAN LB 15-T BR 152DK Functional coating, lamination transparent 15 µm strength breathable Suitable for dry lamination LAMETHAN LB 25 BR 155 Functional coating, lamination transparent 25 µm strength Suitable for dry lamination Good washing stability at 40°C LAMETHAN LB 25 W BR 152 Lamination 25 µm strength breathable Suitable for dry lamination Good washing stability at 40°C LAMETHAN TAPE DE 80 Functional coating, lamination Polymer matrix: polyurethane transparent Good washing stability at 40°C Tape for seam sealing LAMETHAN TAPE ME 160 Functional coating, lamination Polymer matrix: polyurethane transparent Good washing stability at 40°C Tape for seam sealing LAMETHAN VL-H920 O BR150 Functional coating, lamination Two coating layers: membrane and PES satin breathable Suitable for dry lamination Good washing stability at 40°C LAMETHAN VL-H920 S BR 150 Functional coating, lamination Two coating layers: membrane and PES satin breathable Suitable for dry lamination Good washing stability at 40°C LAMETHAN VL-H920 W BR150 Functional coating, lamination Two coating layers: membrane and PES satin breathable Suitable for dry lamination Good washing stability at 40°C TUBICOAT A 12 E Adhesives, functional coatings anions liquid Formaldehyde-free Polymer matrix: polyacrylate TUBICOAT A 17 Adhesives, functional coatings Suitable for tablecloth coating anions liquid Self-crosslinking TUBICOAT A 19 Adhesives, functional coatings Washable anions Formaldehyde-free Good washing stability TUBICOAT A 22 Adhesives, functional coatings Washable middle dura mater anions liquid TUBICOAT A 23 Adhesive middle dura mater anions liquid Applications for changing the feel TUBICOAT A 28 Adhesives, functional coatings anions liquid Formaldehyde-free Good washing stability TUBICOAT A 36 Adhesives, functional coatings Washable anions liquid Low formaldehyde TUBICOAT A 37 Adhesives, functional coatings Washable Suitable for tablecloth coating anions liquid TUBICOAT A 41 Adhesives, functional coatings anions liquid Self-crosslinking Good fastness TUBICOAT A 61 Adhesives, functional coatings Suitable for tablecloth coating liquid Non-ionic Self-crosslinking TUBICOAT A 94 Adhesives, functional coatings anions liquid Self-crosslinking Good fastness TUBICOAT AIB 20 Fashion coating transparent Suitable for foam coatings Pearlescent paint TUBICOAT AOS Foaming aid Non-ionic Foaming Suitable for fluorocarbon coating TUBICOAT ASK Functional coating, lamination Adhesive additives for bonding transparent Suitable for paste coatings Suitable for dry lamination TUBICOAT BH Adhesives, functional coatings Polymer matrix: styrene butadiene anions liquid Formaldehyde-free TUBICOAT B 45 Adhesives, functional coatings Washable Polymer matrix: styrene butadiene anions liquid TUBICOAT BO-NB Functional coating Medium hardness Suitable for light-shielding coatings Good flexibility at low temperatures Suitable for stable foam coatings TUBICOAT BO-W Functional coating Suitable for light-blocking coatings opaque Suitable for stable foam coatings Water vapor permeable TUBICOAT BOS Foaming aid anions Foaming Foam stabilizer TUBICOAT DW-FI Functional coatings, special products anions Suitable for ointment Suitable for stable foam Foamable TUBICOAT E 4 Adhesive anions Self-crosslinking Low formaldehyde Polymer matrix: polyethylene vinyl acetate TUBICOAT ELC Functional coating Suitable for paste coatings black Conductive soft TUBICOAT EMULGATOR HF Functional coatings, special products anions dispersion Suitable for ointment Suitable for stable foam TUBICOAT ENTSCHÄUMER N Defoamers and deaerators liquid Non-ionic Silicon-free Suitable for ointment TUBICOAT FIX FC Fixative cations Water-based liquid Formaldehyde-free TUBICOAT FIX ICB CONC. Fixative liquid Non-ionic Formaldehyde-free Suitable for cross-linking TUBICOAT FIXIERER AZ Fixative liquid Suitable for cross-linking Polyethylenimine-based Uncapped TUBICOAT FIXIERER FA Fixative anions Water-based liquid Low formaldehyde TUBICOAT FIXIERER H 24 Fixative anions Water-based liquid Formaldehyde-free TUBICOAT FIXIERER HT Fixative Water-based liquid Non-ionic Suitable for cross-linking TUBICOAT FOAMER NY Foaming aid Non-ionic Foaming Suitable for fluorocarbon coating No yellowing TUBICOAT GC PU Fashion coating Washable Soft feel Polymer matrix: polyurethane transparent TUBICOAT GRIP Functional coating Anti-slip Suitable for stable foam coatings soft TUBICOAT HEC thickener powder Non-ionic Stable electrolyte Stable against shear forces TUBICOAT HOP-S Special Products anions Suitable for ointment painting Adhesion promoter TUBICOAT HS 8 Adhesive anions liquid Formaldehyde-free dura mater TUBICOAT HWS-1 Functional coating Suitable for paste coatings water proof Suitable for giant umbrellas and tents TUBICOAT KL-TOP F Fashion coating, functional coating Washable Polymer matrix: polyurethane transparent Suitable for paste coatings TUBICOAT KLS-M Fashion coating, functional coating Washable Soft feel Polymer matrix: polyurethane breathable TUBICOAT MAF Fashion coating Washable matrix effect Improve rubbing fastness Soft feel TUBICOAT MD TC 70 Fashion coating Retro wax Suitable for foam coatings Suitable for top coating TUBICOAT MEA Functional coating Washable Polymer matrix: polyurethane Suitable for paste coatings Suitable for top coating TUBICOAT MG-R Fashion coating Washable Soft feel Suitable for paste coatings Double-layer leather finish TUBICOAT MOP NEU Functional coatings, special products Washable anions Foamable paint TUBICOAT MP-D Fashion coating, functional coating Washable Soft feel Medium hardness breathable TUBICOAT MP-W Functional coating Washable Polymer matrix: polyurethane breathable water proof TUBICOAT NTC-SG Functional coating Washable transparent Suitable for paste coatings Medium hardness TUBICOAT PERL A22-20 Fashion coating Suitable for paste coatings Suitable for foam coatings Pearlescent paint TUBICOAT PERL HS-1 Functional coating Suitable for paste coatings Suitable for light-blocking coatings Suitable for pearlescent coatings Suitable for top coating TUBICOAT PERL PU SOFT Fashion coating Washable Golden Turtle Effect Soft feel Polymer matrix: polyurethane TUBICOAT PERL VC CONC. Fashion coating, functional coating Soft feel Polymer matrix: polyurethane Suitable for paste coatings Suitable for light-blocking coatings TUBICOAT PHV Functional coating Medium hardness Suitable for 3D dot painting TUBICOAT PSA 1731 Functional coating, lamination transparent Suitable for paste coatings Suitable for dry lamination Not breathable TUBICOAT PU-UV Adhesive anions liquid Formaldehyde-free Good fastness TUBICOAT PU 60 Adhesive anions liquid Applications for changing the feel Formaldehyde-free TUBICOAT PU 80 Adhesives, functional coatings Washable anions liquid Washable TUBICOAT PUH-BI Adhesive anions liquid Formaldehyde-free dura mater TUBICOAT PUL Functional coating Polymer matrix: polyurethane Suitable for paste coatings Suitable for 3D dot painting Anti-slip TUBICOAT PUS Adhesives, functional coatings anions liquid Formaldehyde-free Polymer matrix: polyurethane TUBICOAT PUW-M Adhesive middle dura mater anions liquid Formaldehyde-free TUBICOAT PUW-S Adhesive anions liquid Formaldehyde-free Good washing stability TUBICOAT PW 14 Adhesives, functional coatings anions Formaldehyde-free Heat sealable Non-wet TUBICOAT SA-M Functional coating Washable Suitable for paste coatings Suitable for 3D dot painting TUBICOAT SCHÄUMER HP Foaming aid, functional coating Non-ionic Foaming Suitable for fluorocarbon coating TUBICOAT SF-BASE Fashion coating Washable Soft feel Suitable for foam coatings Silky sheen effect TUBICOAT SHM Foaming aid anions Foam stabilizer TUBICOAT SI 55 Special Products False cations Suitable for ointment Foamable painting TUBICOAT STABILISATOR RP Foaming aid anions Foam stabilizer TUBICOAT STC 100 Fashion coating, functional coating transparent breathable Suitable for stable foam coatings TUBICOAT STC 150 Fashion coating, functional coating Washable Soft feel transparent breathable TUBICOAT STL Functional coating Washable Anti-slip Suitable for stable foam coatings soft TUBICOAT TCT Fashion coating, functional coating Washable Polymer matrix: polyurethane transparent Suitable for paste coatings TUBICOAT VA 10 Adhesive anions liquid Formaldehyde-free dura mater TUBICOAT VCP Functional coating Suitable for paste coatings Medium hardness Suitable for light-blocking coatings TUBICOAT VERDICKER 17 thickener anions High efficiency synthesis TUBICOAT VERDICKER ASD thickener anions Rapid expansion Stable against shear forces Pseudoplasticity TUBICOAT VERDICKER LP thickener anions Stable against shear forces Pseudoplasticity Dispersible TUBICOAT VERDICKER PRA thickener anions liquid Stable electrolyte Rheological additives TUBICOAT WBH 36 Special Products paint Application to prevent roll deposits TUBICOAT WBV Special Products Non-ionic paint Application to prevent roll deposits TUBICOAT WEISS EU Functional coatings, special products Suitable for ointment Suitable for stable foam Suitable for top coating Titanium dioxide paste TUBICOAT WLI-LT KONZ Functional coating Washable Suitable for paste coatings Anti-slip soft TUBICOAT WLI Fashion coating, functional coating Washable Golden Turtle Effect Soft feel Suitable for paste coatings TUBICOAT WOT Fashion coating Washable Soft feel Suitable for paste coatings Faded effect TUBICOAT WX-TCA 70 Fashion coating, functional coating Retro wax Suitable for paste coatings Suitable for top coating TUBICOAT WX BASE Fashion coating Retro wax Soft feel Suitable for paste coatings Application in primer TUBICOAT ZP NEU Water/oil repellency Zirconium-wax based Suitable for water systems cations Foamable TUBIGUARD 10-F Water/oil repellency Washable Sprayable cations liquid TUBIGUARD 21 Water/oil repellency Washable cations Efficient Water-based TUBIGUARD 25-F Water/oil repellency Washable Sprayable cations Efficient TUBIGUARD 270 Functional coating, water/oil repellency Washable cations Efficient liquid TUBIGUARD 30-F Water/oil repellency Washable Sprayable cations Efficient TUBIGUARD 44 N Water/oil repellency Washable Sprayable Suitable for water systems liquid TUBIGUARD 44N-F Water/oil repellency Suitable for water systems Non-ionic Suitable for polyester Foamable TUBIGUARD 66 Water/oil repellency Washable Sprayable Efficient liquid TUBIGUARD 90-F Water/oil repellency Washable cations Efficient liquid TUBIGUARD AN-F Water/oil repellency Washable Sprayable cations Efficient TUBIGUARD FA2-F Water/oil repellency Sprayable cations Suitable for polyester Foamable TUBIGUARD PC3-F Functional coating, water/oil repellency Washable cations liquid paste TUBIGUARD SR 2010-FW Water/oil repellency cations Efficient Foamable Based on C6 fluorocarbon In some embodiments, the chemicals may include the following, which are supplied by CHT Bezema and are associated with certain selected leather or leather product properties and can be used to enhance the binding of SFS to inkjet printing dyes: CHT-ALGINAT MVU Inkjet printing agents, thickeners cations powder anions High color brightness PRISULON CR-F 50 Inkjet printing agents, thickeners liquid Good outline High surface flatness Good penetration TUBIJET DU 01 Inkjet printing agents Anti-migration agents anions liquid Formaldehyde-free TUBIJET NWA Inkjet printing agents liquid Non-ionic Does not affect the feel Formaldehyde-free TUBIJET PUS Inkjet printing agents Film formation anions liquid Formaldehyde-free TUBIJET VDK Inkjet printing agents liquid Formaldehyde-free Halogen-free Fire protection effect TUBIJET WET Inkjet printing agents anions liquid Does not affect the feel Formaldehyde-free In some embodiments, the chemicals disclosed herein may include the following inkjet printing dyes, which are supplied by CHT Bezema and are associated with certain selected leather or leather product properties and can be used in combination with SFS: BEZAFLUOR BLUE BB pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR GREEN BT pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR ORANGE R pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR PINK BB pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR RED R pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR VIOLET BR pigments High performance BEZAFLUOR (fluorescent pigment) BEZAFLUOR YELLOW BA pigments High performance BEZAFLUOR (fluorescent pigment) BEZAPRINT BLACK BDC pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLACK DT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLACK DW pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLACK GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT BLUE BN pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLUE BT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLUE GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT BLUE RR pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLUE RT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLUE RTM pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BLUE TB pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BORDEAUX K2R pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BROWN RP pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT BROWN TM pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT CITRON 10G pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT CITRON GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT GREEN 2B pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT GREEN BS pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT GREEN BT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT GREY BB pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT NAVY GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT NAVY RRM pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT NAVY TR pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT OLIVE GREEN BT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT ORANGE 2G pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT ORANGE GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT ORANGE GT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT ORANGE RG pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT PINK BW pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT RED 2BN pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT RED GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT RED KF pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT RED KGC pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT SCARLET GRL pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT SCARLET RR pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT TURQUOISE GT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT VIOLET FB pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT VIOLET KB pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT VIOLET R pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT VIOLET TN pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT YELLOW 2GN pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT YELLOW 3GT pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT YELLOW 4RM pigments Advanced BEZAPRINT (classic pigment) BEZAPRINT YELLOW GOT pigments High performance BEZAKTIV GOT (GOTS) BEZAPRINT YELLOW RR pigments Advanced BEZAPRINT (classic pigment)

In some embodiments, the chemicals disclosed herein may include the following, which are supplied by Lamberti SPA and are associated with certain selected leather or leather product properties, which can be used to enhance the adhesion of SFS to painted or repaired surfaces, or SFS can be used to enhance the properties of such chemicals: Before treatment: Aqueous polyurethane dispersion Rolflex AFP. Aliphatic polyether polyurethane aqueous dispersion. This product has high hydrolysis resistance, good breaking load and excellent tear resistance. Rolflex ACF. Aliphatic polycarbonate polyurethane aqueous dispersion. This product exhibits good PU and PVC bonding properties, excellent abrasion resistance and chemical resistance, including alcohol. Rolflex V 13. Aliphatic polyether/acrylic copolymer polyurethane aqueous dispersion. This product has good hot bonding properties and good adhesion to PVC. Rolflex K 80. Aqueous dispersion of aliphatic polyether/acrylic copolymer polyurethane. Rolflex K 80 is specifically designed as a high-performance adhesive for textile lamination. It exhibits excellent perchloroethylene and water fastness. Rolflex ABC. Aqueous dispersion of aliphatic polyether polyurethane. Exceptionally, it exhibits a very high water column, excellent electrolyte resistance, a high LOI index, and high resistance to repeated flexing. Rolflex ADH. Aqueous dispersion of aliphatic polyether polyurethane. It exhibits extremely high water column resistance. Rolflex W4. Aqueous aliphatic polyurethane dispersion, particularly recommended for the formulation of textile coatings for clothing and outerwear requiring a full, soft, and non-tacky feel. Rolflex ZB7. An aliphatic, aqueous polyurethane dispersion particularly recommended for the formulation of textile coatings for technical articles used in apparel, outerwear, sportswear, fashion, and industrial applications. It exhibits very high charge absorption characteristics, electrolyte stability, and excellent mechanical and tear resistance. It is also suitable for foam coatings and dyeing applications. Rolflex BZ 78. An aliphatic, aqueous polyurethane dispersion particularly recommended for the formulation of textile coatings for technical articles used in apparel, outerwear, sportswear, fashion, and industrial applications. It exhibits excellent hydrolysis resistance, very high charge absorption and electrolyte stability, as well as excellent mechanical and tear resistance. It is also suitable for foam coatings and dyeing applications. Rolflex PU 147. An aqueous dispersion of an aliphatic polyether polyurethane. This product exhibits good film-forming properties at room temperature. It has high light and UV fastness and good resistance to water, solvents, chemicals, and mechanical agents. Rolflex SG. An aqueous dispersion of an aliphatic polyether polyurethane. Due to its thermoplastic properties, it is recommended to formulate heat-activated adhesives at low temperatures. Elafix PV 4. An aliphatic blocked isocyanate nanodispersion used to impart anti-shrinkage and anti-pilling properties to pure wool fabrics and their blends. Rolflex C 86. Aliphatic cationic aqueous polyurethane dispersion particularly recommended for the formulation of textile coatings for clothing, outerwear and fashion products requiring a medium softness and a pleasant all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Rolflex CN 29. Aliphatic cationic aqueous polyurethane dispersion particularly recommended for the formulation of textile coatings for clothing, outerwear and fashion products requiring a medium softness and a pleasant all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Oil and water repellents Lamgard FT 60. General-purpose fluorocarbon resin for water and oil repellency; applied by exhaust dyeing. Lamgard 48. High-performance fluorocarbon resin for water and oil repellency; applied by impregnation. High rubbing fastness. Imbitex NRW3. Wetting agent for water and oil repellent finishes. Lamgard EXT. Crosslinker for fluorocarbon resins to improve wash fastness. Flame Retardant Piroflam 712. Non-permanent flame retardant compound for impregnation and spray application. Piroflam ECO. Halogen-free flame retardant compound for back coating of all types of fibers. Piroflam UBC. Flame retardant compound for back coating of all types of fibers. Crosslinker Rolflex BK8. Aqueous dispersion of aromatic-terminated polyisocyanate. It is recommended as a crosslinker in polyurethane-based coating pastes to improve wash fastness. Fissativo 05. Water-dispersible aliphatic polyisocyanate suitable for use as a crosslinker in acrylic and polyurethane dispersions to improve adhesion and wet and dry scrub resistance. Resina MEL. Melamine-formaldehyde resin. Cellofix VLF. Low-formaldehyde melamine resin. Thickener: Lambicol CL 60. Fully neutralized synthetic thickener for pigment printing in oil/water emulsions; medium viscosity type: Viscolam PU conc. Non-ionic polyurethane thickener with pseudoplastic behavior: Viscolam 115 new. Unneutralized acrylic thickener: Viscolam PS 202. Non-ionic polyurethane thickener with Newtonian behavior: Viscolam 1022. Non-ionic polyurethane thickener with medium pseudoplastic behavior. Dyeing Dispersants: Lamegal BO. Non-ionic liquid dispersant suitable for direct, reactive, and disperse dyeing, as well as PES stripping. Lamegal DSP. Dispersing/anti-backstaining agent for the preparation of dyed and printed materials, dyeing, and soaping. Anti-oligomer agent. Lamegal 619. Lamegal TL5, a high-efficiency, low-foaming dispersing and leveling agent for PES dyeing. Lamegal A 12, a multi-purpose chelating and dispersing leveling agent for various textile processes. Lamfix L, a leveling and fixing agent for dyeing wool, polyamide, and their blends with acid or metal complex dyes. Lamfix LU, a formaldehyde-free cationic fixative for direct and reactive dyes. It does not affect color tone or lightfastness. Lamfix PA/TR, a fixing agent for improving the wet fastness of acid dyes on polyamide fabrics, dyeing or printing, and polyamide yarn. Retarding agent for the dyeing of polyamide/cellulose blends with direct dyes. Specialty resin Denifast TC. Specialty resin for the anionization of cellulose fibers to achieve special effects ("DENIFAST system" and "DENISOL system"). Cobral DD/50. Specialty resin for the anionization of cellulose fibers to achieve special effects ("DENIFAST system" and "DENISOL system"). Antireducing agent Lamberti Redox L2S gra. Antireducing agent in granular form. 100% active Lamberti Redox L2S liq. Liquid antireducing agent for automatic dosing. Lubisol AM anti-wrinkle agent . A lubricant and anti-wrinkle agent for wet handling of various fibers and ropes on machinery. Neopat Compound 96/m conc. Pigment and dye migration inhibitor. A compound developed as a migration inhibitor for continuous pigment dyeing processes (dip-drying processes). Neopat Binder PM/S conc. A concentrated form of a specific binder used for preparing dyeing solutions (dip-drying processes) with pigments. Neopat Compound PK1. A highly concentrated all-in-one colorant compound specifically developed as a migration inhibitor and specific binder for continuous pigment dyeing (dip-drying). Neopat Compound FTN. A highly concentrated complex of surfactants and polymers specifically developed for pigment dyeing and pigment-reactive dyeing processes; it is particularly suitable for traditional wash- off finishes in medium/dark shades. Cellofix ULF Conc. An anti-wrinkle modified glyoxylate resin for finishing cotton, cellulose products, and blends with synthetic fibers. Poliflex PO 40. A polyethylene resin with a waxy, full, and smooth feel for application by roll-dyeing. Rolflex WF. An aliphatic, aqueous Nano-PU dispersion used as an extender for wrinkle-free treatments. Texamina C/FPN. A cationic softener with a very soft hand, particularly recommended for exhaust dyeing on all types of fabrics. Also suitable for cone application. Texamina C SAL Flakes. A 100% cationic softener in flake form for all types of fabrics. Dispersible at room temperature. Texamina CL LIQ. An amphoteric softener for all types of fabrics. Non-yellowing. Texamina HVO. An amphoteric softener for knitting and knitting fabrics made of cotton, other cellulose materials, and blends. Provides a soft, smooth, and dry feel. Applied by exhaust dyeing. Texamina SIL. Non-ionic silicone dispersion. Excellent softening, lubricating, and anti-static properties for exhaust dyeing of all fiber types. Texamina SILK. Special cationic softener with silk protein. Provides a "swelling touch" and is particularly suitable for cellulose, wool, and silk. Lamfinish LW. All-purpose complex based on special polymer hydrophilic softeners; applied by painting, roll-on dyeing, and exhaust dyeing. Elastolam E50. Universal one-component silicone elastic softener for textile finishing. Elastolam EC 100. A modified polysiloxane microemulsion that provides a long-lasting finish with an extremely soft, silky feel. Poliflex CSW. Cationic anti-slip agent . Poliflex R 75. A wax finish that imparts a waxy feel. Poliflex S. A compound specially developed for special writing effects. Poliflex M. A compound for a special dry waxy feel. Lamsoft SW 24. A compound specially developed for coating applications with a special slippery feel. Lamfinish SLIPPY. An all-in-one compound that imparts a slippery feel upon application. Lamfinish GUMMY. An all-in-one compound that imparts a jelly-like feel upon application. Lamfinish OLDRY. An all-in-one compound that imparts a dry, sandy feel upon application, particularly suitable for vintage effects. Rolflex LB 2. An aliphatic, water-based polyurethane dispersion , particularly recommended for textile coatings requiring a glossy and stiff top finish. It is particularly suitable as a transparent, gauze-like finish on silk fabrics. Transparent and glossy. Rolflex HP 51. An aliphatic, water-based polyurethane dispersion, particularly recommended for the formulation of textile coatings for outerwear, luggage, and technical articles, especially where a hard yet pliable feel is required. Transparent and glossy. Rolflex PU 879. An aliphatic, water-based polyurethane dispersion, particularly recommended for the formulation of textile coatings for outerwear, luggage, and technical articles requiring a medium hardness yet a pliable feel. Rolflex ALM. An aliphatic, water-based polyurethane dispersion, particularly recommended for the formulation of textile coatings for outerwear, luggage, and technical articles requiring a soft and pliable feel. Also suitable for dyeing and printing applications. Rolflex AP. This aliphatic, water-based polyurethane (PU) dispersion is particularly recommended for the formulation of textile coatings for outerwear and fashion products requiring a soft, colloid feel. Rolflex W4. This aliphatic, water-based polyurethane (PU) dispersion is particularly recommended for the formulation of textile coatings for apparel and outerwear requiring a full, soft, and non-tacky feel. Rolflex ZB7. This aliphatic, water-based polyurethane (PU) dispersion is particularly recommended for the formulation of textile coatings for apparel, outerwear, sportswear, fashion, and technical products for industrial applications. This product exhibits very high charge absorption characteristics, electrolyte stability, and excellent mechanical and tear resistance. It is also suitable for foam coatings and printing and dyeing applications. Rolflex BZ 78. This aliphatic, water-based polyurethane dispersion is particularly recommended for the formulation of textile coatings for use in clothing, outerwear, sportswear, fashion, and technical articles for industrial applications. It exhibits excellent resistance to hydrolysis, very high charge absorption and electrolyte stability, and excellent mechanical and tear resistance. It is also suitable for foam coatings and printing and dyeing applications. Rolflex K 110. It imparts a full, soft, and slightly tacky feel to coated fabrics and offers excellent fastness properties on all fabric types. Rolflex OP 80. Aliphatic water-based polyurethane dispersion, particularly recommended for the formulation of textile coatings for outerwear, luggage and fashion finishes requiring an opaque, non-writing effect. Rolflex NBC. Aliphatic water-based polyurethane dispersion, typically used as a filler and formaldehyde-free sizing agent for exhaust dyeing applications. Can be used for outerwear and fashion finishes requiring a full, elastic and non-tacky feel. Rolflex PAD. Aliphatic water-based polyurethane dispersion, specifically designed for exhaust dyeing applications for outerwear, sportswear and fashion applications requiring a full, elastic and non-tacky feel. Excellent wash and dry cleaning fastnesses and good bath stability. Rolflex PN. An aliphatic, water-based polyurethane dispersion, typically used by exhaust application for outerwear and high-quality fashion applications requiring a strong, elastic, non-sticky finish. Elafix PV 4. An aliphatic, blocked isocyanate nanodispersion, used to impart anti-shrinkage and anti-pilling properties to pure wool fabrics and their blends. Rolflex SW3. An aliphatic, water-based polyurethane dispersion, particularly recommended by exhaust application for outerwear, sportswear, and fashion finishes requiring a smooth and elastic feel. It is also a good anti-pilling agent. It performs particularly well in wool applications. Rolflex C 86. An aliphatic, cationic, aqueous polyurethane dispersion particularly recommended for use in textile coatings for apparel, outerwear, and fashion products requiring a medium softness and a comfortable all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Rolflex CN 29. An aliphatic, cationic, aqueous polyurethane dispersion particularly recommended for use in textile coatings for apparel, outerwear, and fashion products requiring a softness and a comfortable all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Other resins include: Textol 110, a soft feel modifier for textile coatings; Textol RGD, an aqueous emulsion of acrylic copolymers for textile coatings, with a very firm feel; Textol SB 21, a butadiene resin for coatings and a binder for textile printing and dyeing; Appretto PV/CC, an aqueous dispersion of vinyl acetate for stiffening; Amisolo B, an aqueous dispersion of CMS for textile coatings ; Lamovil RP, a stabilized solution of PVOH for stiffening; and Rolflex AFP, an aqueous dispersion of aliphatic polyether polyurethane . This product has high hydrolysis resistance, good breaking load and excellent tear resistance. Rolflex ACF. Aliphatic polycarbonate polyurethane aqueous dispersion. This product shows good PU and PVC bonding properties, excellent abrasion resistance and chemical resistance, including alcohols. Rolflex V 13. Aliphatic polyether/acrylic copolymer polyurethane aqueous dispersion. This product has good heat bonding properties and good adhesion to PVC. Rolflex K 80. Aliphatic polyether/acrylic copolymer polyurethane aqueous dispersion. ROLFLEX K 80 is specially designed as a high-performance adhesive for textile lamination. This product has excellent perchloroethylene and water fastness. Rolflex ABC. Aliphatic polyether polyurethane aqueous dispersion. In particular, this product exhibits a very high water column, excellent electrolyte resistance, a high LOI index, and high resistance to repeated bending. Rolflex ADH. Aliphatic polyether polyurethane aqueous dispersion. This product has an extremely high water column resistance. Rolflex W4. Aliphatic aqueous polyurethane dispersion, particularly recommended for the formulation of textile coatings for clothing and outerwear that require a saturated, soft, and non-sticky feel. Rolflex ZB7. Aliphatic aqueous polyurethane dispersion, particularly recommended for the formulation of textile coatings for clothing, outerwear, sportswear, fashion and technical articles for industrial applications. This product has very high charge absorption characteristics, electrolyte stability, and excellent mechanical and tear resistance. Also suitable for foam coating and printing and dyeing applications. Rolflex BZ 78. Aliphatic aqueous PU dispersion, particularly recommended for formulating textile coatings for technical articles for clothing, outerwear, sportswear, fashion and industrial applications. The product has excellent resistance to hydrolysis, very high charge absorption and electrolyte stability, as well as excellent mechanical and tear resistance. Also suitable for foam coating and printing and dyeing applications. Rolflex PU 147. Aliphatic polyether polyurethane aqueous dispersion. This product shows good film-forming properties at room temperature. It has high light and UV fastness and good resistance to water, solvents and chemicals, as well as mechanical resistance. Rolflex SG. Aliphatic polyether polyurethane aqueous dispersion. Due to its thermoplastic properties, it is recommended to formulate heat-activated adhesives at low temperatures. Elafix PV 4. Aliphatic blocked isocyanate nanodispersion used to impart anti-shrinkage and anti-pilling properties to pure wool fabrics and their blends. Rolflex C 86. Aliphatic cationic aqueous polyurethane dispersion, particularly recommended for formulating textile coatings for apparel, outerwear, and fashion products requiring a medium softness and comfortable all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Rolflex CN 29. Aliphatic cationic aqueous polyurethane dispersion, particularly recommended for coating textiles for apparel, outerwear, and fashion that require a soft and comfortable all-around feel. Fabrics treated with this product can be dyed with a range of dyes to achieve two-tone effects of varying intensities. Oil and water repellents Lamgard FT 60. General-purpose fluorocarbon resin for water and oil repellency; applied by exhaust dyeing. Lamgard 48. High-performance fluorocarbon resin for water and oil repellency; applied by exhaust dyeing. High rubbing fastness. Imbitex NRW3. Wetting agent for water and oil repellent coatings. Lamgard EXT. Crosslinking agent for fluorocarbon resins to improve wash fastness. Flame retardant Piroflam 712. Non-permanent flame retardant compound for impregnation and spray application. Piroflam ECO. Halogen-free flame retardant compound for all types of fiber back coating applications. Piroflam UBC. Flame retardant compound for all types of fiber back coating applications. Crosslinker Rolflex BK8. Aqueous dispersion of aromatic terminated polyisocyanate. Recommended as a crosslinker in polyurethane resin-based coating pastes to improve wash fastness. Fissativo 05. Water-dispersible aliphatic polyisocyanate suitable as a crosslinker for acrylic and polyurethane dispersions to improve adhesion and wet and dry scrub resistance. Resina MEL. Melamine-formaldehyde resin. Cellofix VLF. Low-formaldehyde melamine resin. Thickener: Lambicol CL 60. Fully neutralized synthetic thickener for pigment printing in oil/water emulsions; medium viscosity type: Viscolam PU conc. Non-ionic polyurethane-based thickener with pseudoplastic behavior: Viscolam 115 new. Unneutralized acrylic thickener: Viscolam PS 202. Non-ionic polyurethane-based thickener with Newtonian behavior: Viscolam 1022. Non-ionic polyurethane-based thickener with medium pseudoplastic behavior.

In some embodiments, the chemical agent may include one or more of silicone, acidic agents, dyes, pigments, traditional coatings, and technical coatings. Dyes may include one or more of dispersants, leveling agents, fixatives, specialized resins, anti-reduction agents, and anti-wrinkle agents. Pigments may include one or more of anti-migration agents, adhesives, all-in-one agents, and rendering agents. Traditional coatings may include one or more of wrinkle-free treatment agents, softeners, feel modifiers, water-based polyurethane dispersions, and other resins. The technical coating agent may include one or more of an aqueous polyurethane dispersion, an oil repellent, a water repellent, a crosslinking agent, and a thickener.

In some embodiments, certain chemicals disclosed herein can be provided by one or more of the following chemical suppliers: Adrasa, AcHitex Minerva, Akkim, Archroma, Asutex, Avocet dyes, BCC India, Bozzetto group, CHT, Clariant, Clearity, Dilube, Dystar, Eksoy, Erca group, Genkim, Giovannelli e Figli, Graf Chemie, Huntsman, KDN Bio, Lamberti, LJ Specialties, Marlateks, Montegauno, Protex, Pulcra Chemicals, Ran Chemicals, Fratelli Ricci, Ronkimya, Sarex, Setas, Silitex, Soko Chimica, Tanatex Chemicals, Union Specialties, Zaitex, Zetaesseti, and Z Schimmer.

In some embodiments, the chemical agent may include an acidic agent. Thus, in some embodiments, the SFS may include an acidic agent. In some embodiments, the acidic agent may be Bronsted acid. In one embodiment, the acidic agent includes one or more of citric acid and acetic acid. In one embodiment, the acidic agent facilitates the deposition and application of the SPF mixture (i.e., the SFS coating) onto the leather or leather product to be coated, compared to the absence of such an acidic agent. In one embodiment, the acidic agent improves crystallization of the SPF mixture onto the textile to be coated.

In one embodiment, the acidic agent is present in an amount greater than about 0.001%, or greater than about 0.002%, or greater than about 0.003%, or greater than about 0.004%, or greater than about 0.005%, or greater than about 0.006%, or greater than about 0.007%, or greater than about 0.008%, or greater than about 0.009%, or greater than about 0.01%, or greater than about 0.02%, or greater than about 0.03%, or greater than about 0.04%, or greater than about 0.05%, or greater than about 0.06%, or greater than about 0.07%, or greater than about 0.08%, or greater than about 0.09%, or greater than about 0.1%, or greater than about 0.2%, or greater than about 0.3%, or greater than about 0.4%, or greater than about 0.5%, or greater than about 0.6%, or greater than about 0.7%, or greater than about 0.8%, or greater than about 0.9%, or greater than about 1.0%, or greater than about 2.0%, or greater than about 3.0%, or greater than about 4.0%, or greater than about 5.0%.

In one embodiment, the acidic agent is present in an amount by weight (% w/w or % w/v) or by volume (v/v) of less than about 0.001%, or less than about 0.002%, or less than about 0.003%, or less than about 0.004%, or less than about 0.005%, or less than about 0.006%, or less than about 0.007%, or less than about 0.008%, or less than about 0.009%, or less than about 0.01%, or less than about 0.02%, or less than about 0.03%, or less than about 0.04%, or less than about 0.05%, or less than about 0.06%, or less than about 0.07%, or less than about 0.08%, or less than about 0.09%, or less than about 0.1%, or less than about 0.2%, or less than about 0.3%, or less than about 0.4%, or less than about 0.5%, or less than about 0.6%, or less than about 0.7%, or less than about 0.8%, or less than about 0.9%, or less than about 1.0%, or less than about 2.0%, or less than about 3.0%, or less than about 4.0%, or less than about 5.0%.

In some embodiments, the SFS may have a pH of less than about 9, or less than about 8.5, or less than about 8, or less than about 7.5, or less than about 7, or less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4, or greater than about 3.5, or greater than about 4, or greater than about 4.5, or greater than about 5, or greater than about 5.5, or greater than about 6, or greater than about 6.5, or greater than about 7, or greater than about 7.5, or greater than about 8, or greater than about 8.5.

In some embodiments, the SFS may include an acidic agent and may have a pH of less than about 9, or less than about 8.5, or less than about 8, or less than about 7.5, or less than about 7, or less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4, or greater than about 3.5, or greater than about 4, or greater than about 4.5, or greater than about 5, or greater than about 5.5, or greater than about 6, or greater than about 6.5, or greater than about 7, or greater than about 7.5, or greater than about 8, or greater than about 8.5.

In one embodiment, the chemical agent may include silicone. In some embodiments, the SFS may include silicone. In some embodiments, the leather or leather product may be pre-treated (i.e., before the SFS is applied) or post-treated (i.e., after the SFS is applied) with silicone.

In some embodiments, the silicone may include a silicone emulsion.

The term "polysilicone" generally refers to a broad family of synthetic polymers, polymer blends, and/or emulsions thereof having a repeating silicon-oxygen backbone, including but not limited to polysiloxanes. In some embodiments, the polysilicone may include any of the polysilicone species disclosed herein.

More generally, silicones may be used, for example, to improve the feel, but also to increase the water repellency (or reduce the water transmission properties) of the silicone-coated material.

In some embodiments, the SFS may include a concentration of less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 9%, or less than about 8%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.9%, or less than about 0.8%, or less than about 0.7%, or less than about 0.6%, or less than about 0.5%, or less than about 0.4%, or less than about 0.3%, or less than about 0.2%, or less than about 0.1%, or less than about 0.01%, or less than about 0.001% silicone.

In some embodiments, the SFS may include a concentration of greater than about 25%, or greater than about 20%, or greater than about 15%, or greater than about 10%, or greater than about 9%, or greater than about 8%, or greater than about 7%, or greater than about 6%, or greater than about 5%, or greater than about 4%, or greater than about 3%, or greater than about 2%, or greater than about 1%, or greater than about 0.9%, or greater than about 0.8%, or greater than about 0.7%, or greater than about 0.6%, or greater than about 0.5%, or greater than about 0.4%, or greater than about 0.3%, or greater than about 0.2%, or greater than about 0.1%, or greater than about 0.01%, or greater than about 0.001% silicone.

In some embodiments, the SFS can be provided in a concentrated form suspended in water. In some embodiments, the SFS can have a concentration of less than about 50% by weight (% w/w or % w/v), or less than about 45%, or less than about 40%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.1%, or less than about 0.01%, or less than about 0.001%, or less than about 0.0001%, or less than about 0.00001%. In some embodiments, the SFS may have a concentration of greater than about 50% by weight (% w/w or % w/v), or greater than about 45%, or greater than about 40%, or greater than about 35%, or greater than about 30%, or greater than about 25%, or greater than about 20%, or greater than about 15%, or greater than about 10%, or greater than about 5%, or greater than about 4%, or greater than about 3%, or greater than about 2%, or greater than about 1%, or greater than about 0.1%, or greater than about 0.01%, or greater than about 0.001%, or greater than about 0.0001%, or greater than about 0.00001%.

In some embodiments, the SFS coating may include an SFS as described herein. In some embodiments, the SFS may include silicone and/or an acidic agent. In some embodiments, the SFS may include silicone and an acidic agent. In some embodiments, the SFS may include silicone, an acidic agent, and/or an additional chemical, wherein the additional chemical may be one or more of the chemicals described herein. In some embodiments, the SFS may include a silicone emulsion and an acidic agent, such as acetic acid or citric acid.

In some embodiments, the coating process of the present disclosure may include a finishing step for the resulting coated material. In some embodiments, finishing or final finishing of the material coated with SFS in the process of the present disclosure may include sanding, steaming, brushing, polishing, compacting, napping, grinding, shearing, heat setting, waxing, air spraying, calendering, pressing, shrinking, treating with a polymerizer, painting, laminating, and/or laser etching. In some embodiments, finishing of SFS-coated materials may include processing the textiles with an AIRO® 24 dryer, which can be used for both continuous and open-width tumbling of woven, nonwoven, and knitted fabrics. Coating Performance Testing

In embodiments, the coating system described herein passes a wet colorfastness rub test up to 600 cycles, passes a tape test, and passes a Bally flex test up to 20,000 cycles with no observed delamination. Some non-limiting examples of performance testing are further described below. Veslic Test / Colorfastness Rub Test

Dry rubbing colorfastness refers to the fading and staining resistance of a dyed fabric when rubbed with a cloth, felt, or similar material. Wet rubbing colorfastness refers to the fading and staining resistance of a dyed fabric when rubbed with a cloth, felt, or similar material having a moisture content of 95% to 105%. In one embodiment, the coating system described herein passed the dry CFR test for up to 1,000 cycles with a score of 5. In other words, little or no fading of the leather or staining of the rubbing material was observed over up to 1,000 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 1,000 level 5 cycles, up to 1,000 level 4 cycles, up to 1,000 level 3 cycles, up to 1,000 level 2 cycles, or up to 1,000 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 900 level 5 cycles, up to 900 level 4 cycles, up to 900 level 3 cycles, up to 900 level 2 cycles, or up to 900 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 800 level 5 cycles, up to 800 level 4 cycles, up to 800 level 3 cycles, up to 800 level 2 cycles, or up to 800 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 700 level 5 cycles, up to 700 level 4 cycles, up to 700 level 3 cycles, up to 700 level 2 cycles, or up to 700 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 600 level 5 cycles, up to 600 level 4 cycles, up to 600 level 3 cycles, up to 600 level 2 cycles, or up to 600 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 500 level 5 cycles, up to 500 level 4 cycles, up to 500 level 3 cycles, up to 500 level 2 cycles, or up to 500 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 400 level 5 cycles, up to 400 level 4 cycles, up to 400 level 3 cycles, up to 400 level 2 cycles, or up to 400 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 300 level 5 cycles, up to 300 level 4 cycles, up to 300 level 3 cycles, up to 300 level 2 cycles, or up to 300 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 200 level 5 cycles, up to 200 level 4 cycles, up to 200 level 3 cycles, up to 200 level 2 cycles, or up to 200 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of up to 100 level 5 cycles, up to 400 level 4 cycles, up to 100 level 3 cycles, up to 100 level 2 cycles, or up to 100 level 1 cycles. In embodiments, the coating system described herein passes a dry CFR test of 800-1000 level 5 cycles, 800-1000 level 4 cycles, 800-1000 level 3 cycles, 800-1000 level 2 cycles, or 800-1000 level 1 cycles. In embodiments, the coating system described herein passes a dry CFR test of 600-800 level 5 cycles, 600-800 level 4 cycles, 600-800 level 3 cycles, 600-800 level 2 cycles, or 600-800 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of 500-600 level 5 cycles, 500-600 level 4 cycles, 500-600 level 3 cycles, 500-600 level 2 cycles, or 500-600 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of 400-500 level 5 cycles, 400-500 level 4 cycles, 400-500 level 3 cycles, 400-500 level 2 cycles, or 400-500 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of 500-1000 level 5 cycles, 500-1000 level 4 cycles, 500-1000 level 3 cycles, 500-1000 level 2 cycles, or 500-1000 level 1 cycles. In embodiments, the coating systems described herein pass a dry CFR test of 100-500 level 5 cycles, 100-500 level 4 cycles, 100-500 level 3 cycles, 100-500 level 2 cycles, or 100-500 level 1 cycles.

In embodiments, the coating systems described herein pass a wet CFR test of up to 600 level 5 cycles, up to 600 level 4 cycles, up to 600 level 3 cycles, up to 600 level 2 cycles, or up to 600 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 600 level 5 cycles, up to 600 level 4 cycles, up to 600 level 3 cycles, up to 600 level 2 cycles, or up to 600 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 500 level 5 cycles, up to 500 level 4 cycles, up to 500 level 3 cycles, up to 500 level 2 cycles, or up to 500 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 400 level 5 cycles, up to 400 level 4 cycles, up to 400 level 3 cycles, up to 400 level 2 cycles, or up to 400 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 300 level 5 cycles, up to 300 level 4 cycles, up to 300 level 3 cycles, up to 300 level 2 cycles, or up to 300 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test (also known as a wet Veslic test) of up to 200 level 5 cycles, up to 200 level 4 cycles, up to 200 level 3 cycles, up to 200 level 2 cycles, or up to 200 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 100 level 5 cycles, up to 100 level 4 cycles, up to 100 level 3 cycles, up to 100 level 2 cycles, or up to 100 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 50 level 5 cycles, up to 50 level 4 cycles, up to 50 level 3 cycles, up to 50 level 2 cycles, or up to 50 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of up to 20 Level 5 cycles, up to 20 Level 4 cycles, up to 20 Level 3 cycles, up to 20 Level 2 cycles, or up to 20 Level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of 100-500 Level 5 cycles, 100-500 Level 4 cycles, 100-500 Level 3 cycles, 100-500 Level 2 cycles, or 100-500 Level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of 500-600 level 5 cycles, 500-600 level 4 cycles, 500-600 level 3 cycles, 500-600 level 2 cycles, or 500-600 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of 400-500 level 5 cycles, 400-500 level 4 cycles, 400-500 level 3 cycles, 400-500 level 2 cycles, or 400-500 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of 200-400 level 5 cycles, 200-400 level 4 cycles, 200-400 level 3 cycles, 200-400 level 2 cycles, or 200-400 level 1 cycles. In embodiments, the coating systems described herein pass a wet CFR test of 100-200 level 5 cycles, 100-200 level 4 cycles, 100-200 level 3 cycles, 100-200 level 2 cycles, or 100-200 level 1 cycles. In embodiments, the coating system described herein passes a wet CFR test of 10 to 100 level 5 cycles, 10 to 100 level 4 cycles, 10 to 100 level 3 cycles, 10 to 100 level 2 cycles, or 10 to 100 level 1 cycles.

The Bally flex test is performed to determine the leather's resistance to bending by bending the leather at certain angles and speeds. Samples are loaded into a 12-station Bally flex tester (Schap Specialty Machine) and subjected to flexion cycles. In embodiments, the substrate and coating systems disclosed herein pass Bally flex tests for up to 1,000 cycles, up to 5,000 cycles, up to 10,000 cycles, up to 15,000 cycles, and up to 20,000 cycles without delamination. In other words, there is no separation between the coating system and the substrate. In embodiments, the substrates and coating systems disclosed herein pass the Barre flexion test from 1,000 cycles to 5,000 cycles, from 5,000 cycles to 10,000 cycles, from 10,000 cycles to 15,000 cycles, or from 15,000 cycles to 20,000 cycles without delamination .

In the tape test, a piece of tape (i.e., Scotch tape) is applied to the leather, pressed firmly by hand, then removed and inspected for any particles that have fallen off the leather. If no particles are visible on the tape, it can be inferred that no delamination or separation between the substrate and the coating has been observed. In the examples of this disclosure, no delamination was observed using 4 g/sqft L5267 and 6 g/sqft L0822. Paper Transfer Process

In some embodiments, the layers or coatings described herein can be applied via a paper transfer or paper peeling process. See, for example, Figures 129 and 130 . A description of a paper peeling process can also be found in International Publication No. WO2020209717, which is incorporated herein by reference. The paper peeling process can be used to emboss textured patterns onto substrates (e.g., leather) while maintaining texture integrity and imparting a luxurious quality to the pattern or textile. Example

The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and variations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Example 1: Colorfastness to Rubbing

Color fastness to rubbing (wet Veslic test) is one of the most important and challenging technical specifications for leather finishing chemistries. Here, we demonstrate that the silk fibroin fragment composition described herein (activated silk, entry B2) outperforms a polyurethane system specifically designed as a topcoat (Stahl WT-13-097), exhibiting enhanced CFR performance (Stahl WT-42-518) at lower dry weight loadings deposited on the leather surface. Specifically, the silk fibroin fragment composition was able to withstand >600 rubbing cycles without any degradation of the leather's appearance or water repellency, making it a viable alternative to polyurethane in leather finishing. In terms of CFR requirements for various use cases, the luxury goods industry requires a minimum of 10 cycles, the furniture market requires a minimum of 500 cycles, and the automotive market requires 500-1000 cycles. The performance revealed here shows that the silk fibroin fragment (entry B2) far exceeds the CFR requirements for luxury goods and is an early indicator for the use of Activated Silk™ in use cases such as automotive leather and furniture, where higher performance is required (see Figure 4 for photos of felt pads (and associated leather samples) after 600 consecutive cycles of wet Veslic rubbing, comparing the leather sample treated with the silk fibroin fragment composition (bottom sample - entry B2) to the leather sample treated with polyurethane (top 2 samples). Note the damage to the polyurethane sample and the loss of dye from the leather to the felt after 600 cycles).

Materials: AS™ Formulation: The formulation evaluated consisted of two components that were sequentially deposited onto the surface of leather samples by spray application: Component 1: A silk fibroin fragment composition (Activated Silk™) with 0-5% crosslinking agent in water. Component 2: A proprietary additive delivered in ethanol.

Polyurethane Reference Samples: PU1: Stahl WT-42-518 crosslinked with 5% Melio 09S11. The total solids content of Stahl WT-42-518 is 10%. PU2: Stahl WT-13-097 crosslinked with 5% Melio 09S11. The total solids content of Stahl WT-13-097 is 8.75%.

Leather Sample: Bodin Brown (Shade 872) plongé leather sample obtained from Bodin-Joyeux and used as received. Procedure Coating Process

Components 1 and 2 were delivered sequentially to the leather surface by spray application. The spray was applied from a distance of 2 ft and at an outlet pressure of 60 psi. The wet mass loading of each layer was set to 3 g/ ^ft² and measured directly after deposition. The samples were allowed to dry visually between deposition steps.

PU1 and PU2: Delivered in a single pass using the same spraying method described in this article. Target wet mass loading was 3 g/ ^ft² .

Color fastness to wet Veslic rubbing (ISO 11640): Testing was performed as described in EBN-SOP-TXTL-035. Samples were allowed to stand for 48 hours prior to testing. Results

Initial screening results showed that color fastness to wet Veslic rubbing (10 cycles) was significantly improved when using silk fibroin fragments. The data are summarized in Table 1 and Figure 5. The reproducibility of the solutions is highlighted in Table 2. Table 1 contains the CFR results for various formulations. Figure 5: Photograph of a felt pad after 10 cycles of wet Veslic rubbing on leather samples treated with silk fibroin fragments. Table 2: Reproducibility of color fastness to wet Veslic rubbing (ISO 11640) results for silk fibroin fragments (600 cycles). Figure 5. Photograph of a felt pad after 10 cycles of wet Veslic rubbing on leather samples treated with items A1, A2, B1, and B2 (Table 1). ^a. Cellulose derivatives include methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. ^b. Plasticizers include triethyl citrate, dibutyl sebacate, triacetin, glycerol, 1,3-propylene glycol, propylene glycol, pentylene glycol, epoxidized vegetable oil, isosorbide esters, succinic acid derivatives, and monoglyceride acetate. ^c. Crosslinkers include polyisocyanate, polycarbodiimide, polyaziridine, polyurea, glutaraldehyde, and starch dialdehyde.

Using these results, the color fastness to wet Veslic rubbing was extended from 10 cycles to 600 cycles for the formulation described in entry B2. The results of this experiment are shown in Figure 4. These experiments were repeated using standard crosslinked polyurethane coating systems (PU1 and PU2) for comparison. Figure 4: Comparative photographs of a felt pad and a related leather sample after 600 consecutive cycles of wet Veslic rubbing. Top: Commercial reference (crosslinked polyurethanes PU1 and PU2); Bottom: Fiber protein fragment. Figure 4. Photographs of felt pads (and associated leather samples) after 600 consecutive cycles of wet Veslic abrasion, comparing leather samples treated with a composition of filamentous protein fragments (Activated Silk™) (entry B2) and leather samples treated with PU1 and PU2 (commercial references).

The water repellency of the fibroin fragments is qualitatively depicted in Figure 6. Figure 6 depicts the water repellency of leather treated with the fibroin fragments after 600 cycles of wet Veslic rubbing, compared to 10 cycles for the cross-linked polyurethanes PU1 and PU2. Figure 6. Photographs of a water droplet placed on samples treated with either the fibroin fragment or the cross-linked polyurethane coating system after wet Veslic rubbing. In the case of the fibroin fragment (entry B2), the sample was exposed to 600 rubbing cycles, while the polyurethane sample was subjected to only 10 cycles. The photograph was taken 5 minutes after the water droplet was placed. Please note that when using commercial reference systems designed as top coats, water penetrates into the leather matrix.

The results shown in Figures 4-7 and Tables 1-2 demonstrate a significant improvement in the wet CFR performance of the fibrous protein fragment (entry B2) deposited on Bodin brown leather. This performance was extended to at least 600 consecutive cycles without degradation in wet CFR performance, and was superior to that of conventional cross-linked polyurethane systems.

As shown in Figure 6, the silk fibroin fragments provide enhanced water repellency, which is maintained even after the abrasion encountered during wet Veslic rubbing. Example 2: Coated aniline leather

In some embodiments, the leather coating may contain multiple layers, including an adhesive layer and a top coat, as appropriate. The adhesive layer may contain a bio-derived polyurethane (e.g., Biopur 3015), a fibrous protein fragment composition (e.g., AS-104 LS), as appropriate, and a solvent (e.g., water). The bio-derived polyurethane content may be 20% to 21%, 21% to 22%, 22% to 23%, or 23% to 24%. The fibrous protein fragment composition content may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

The top coating may contain a cellulose derivative, an alcohol solvent, and a glycerol derivative (e.g., glycerol acetone (AUGEO SL 191)). In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. The cellulose derivative content may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The glycerol derivative content may be 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, or 14% to 15%. Suitable solvents include, but are not limited to, methanol, ethanol, acetone, isopropyl alcohol, n-butanol, or combinations thereof. The top coat may contain 40% to 41%, 41% to 42%, 42% to 43%, 43% to 44%, 44% to 45%, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49%, or 49% to 50% ethanol. The top coat may contain 30% to 31%, 31% to 32%, 32% to 33%, 33% to 34%, 34% to 35%, 35% to 36%, 36% to 37%, 37% to 38%, 38% to 39%, or 39% to 40% n-butanol. Without wishing to be bound by any particular theory, it is believed that the solvent used in any layer described herein will provide the greatest benefit during the coating application step and/or process and will thereafter be largely removed during the drying step and/or process. The application rate of each layer on the solid can be from about 0.25 to about 1.5 g/ft ² for the optional adhesive layer, such as, but not limited to, about 0.728 g/ft ² , and from about 0.015 to about 0.15 g/ft ² for the top coat, such as, but not limited to, about 0.05 g/ft ^2. Table 3 below shows non-limiting examples of top coat and adhesive layer compositions. Example 3: Purpose / Scope of Study on Activated Silk on Nubuck Leather

The main objective of this study was to evaluate the potential of a fibroin fragment composition (e.g., 117-AS) as a fixing agent for leather dyes. The idea was to compare the classical fixing agents used in leather dyeing with the fibroin fragment composition and to evaluate the comparative performance and the effect on the leather (color and feel). The fixing agent used as a reference was OPTIFIX E-50 liq, an aliphatic polyamine. Base materials / formulations used

Nubuck leather was used as the substrate for dyeing with DORAN IL ORANGE BROWN S3R, a leather dye with poor rubbing fastnesses, to demonstrate the fastness-improving effects of the fixative. After acidification, the nubuck leather was top-dyed at a rate of 4% plus 2% (based on shaved hair weight). Optifix ES0 was used as the fixative, typically used in the wet-end stage, and was applied as follows: At 40°C: ● 300% water ● 0.2% formic acid ● 10-minute run ● 2% Optifix ES0 ● 20-minute run ● 0.4% formic acid ● Drain and rinse ● Dry until crusty

The activated silk is applied by spraying during the painting stage.

The crosslinking of the activated yarns was carried out using two different systems: a. Using CARTARETIN F liquid, an aqueous solution of polyamide amine used in the paper industry and which has shown excellent results as a crosslinking agent for bio-based acrylics. b. Using MELIO 09-S-11 (Stahl) Additional notes: Optifix E-50 liquid, pH 3.8-4.0, was applied in a separate bath, running for 30 minutes. The amounts in references 2 to 7 are parts per 100. They were all applied by spraying, with two crosses. Results

Pieces treated with different systems were cured during the 24-hand rub test ISO 105-X12.

The results are summarized in the table below: Note: All evaluations are on a scale of 1 to 5, meaning: Change in color/touch: 5 = minimal effect/change Crock fastness: 5 = maximum resistance = minimal staining

In both systems, the improvement in dry rubbing fastness with 117-AS was significant. The improvement in wet rubbing fastness was quite low, and in the case of isocyanates, no improvement at all. The best scores were achieved with a moderate amount of 117-AS in the formulation (approximately 17%). The finishing spray always produced a film-forming effect that was unhelpful on nubuck leather. Example 4: Silk Solution for Leather Treatment

A number of silk solutions were prepared for treating leather, as described in Table 6, and can be used as described herein.

The silk formulations as described herein can be used before, during, or after various leather processing steps, including: Drying - Drying of hand and automatically sprayed hides can be performed in the production line oven used during normal leather processing. Automatically sprayed hides can be dried one or more times between one or more spray treatments, for example, spray > dry > spray > dry. The oven temperature can be varied between 70-75°C, and each drying cycle can last about 25 seconds. Punch Pressing - Punch pressing can be used in the leather production process. In this process, the hide is compressed (treated side up) between two metal plates (approximately 5-6 ^m2 ), with the top plate operating at 57°C. The leather is compressed at this temperature for 2 seconds at 100 kg/ ^cm² . Qualitatively, the pressing process adds a gloss to the leather sample. Finiflex – A typical processing step for plongé leather, this mechanical treatment can be used as the final step for silk-doped leather. On this machine, the leather is processed in half – one half is lifted onto a rotating heated metal wheel (93°C; 20 kg/ ^m² ; d- _wheel = 0.3 m) and compressed for 4 seconds. The leather is then pulled out, turned over, and the other half treated in the same way. Uniflex – The Uniflex treatment is similar to the Finiflex treatment and is used at the end of leather processing. In this process, the hide is fed onto a feed belt into two pressing cylinders (each with a diameter of 0.3 m). The top cylinder is heated to 60°C, while the bottom cylinder is unheated. The cylinders compress the hide together at 30 bar for 3-5 seconds. Polishing - The polisher scrapes off some of the surface treatment applied to the hide in the previous processing steps (physical abrasion). In the early stages of leather processing, this serves to "open" the hide, allowing the fixing agent/pigmentation agent to adhere more effectively in a similar way to the mechanical stretching process that occurs before trimming the hide. Automatic sprayer - Unless otherwise stated, when the hide is sprayed using an in-house automatic sprayer, it can be sprayed in one or more rounds with an intermediate drying treatment. The spray fluid (filament, silicone treatment agent, etc.) can be pumped into the nozzle feed line at 3 bar and fed into the nozzle inlet ( _Dnozzle = 0.6 mm) at a pressure between 0.8 and 1.2 bar. The spray volume of the automatic sprayer can vary from 0.8 to 1.0 g/ ^ft² . The dwell volume of the spray fluid can be approximately 2 to 2.5 L. The various filament formulations described herein can be fed into this type of machine and evenly applied to the skin.

The manual spray process can involve depositing one or more coats of silk on one half of a piece of leather, for example, two passes with different orientations, coat 1 with a vertically oriented spray pattern and coat 2 with a horizontally oriented spray pattern, with the other half covered as a control. The manual spray volume can be about 50 mL per coat.

When placed under observation light, the 6% coated skin may have a noticeably darker sheen and may feel slightly harder to the touch than the untreated control. Example 5: Formulation Preparation

Materials: Selected salts include calcium chloride ( _CaCl₂ ), sodium chloride (NaCl), magnesium sulfate heptahydrate ( _MgSO₄ ), guanidine hydrochloride (GdmCl), L-arginine hydrochloride (ArgCl), urea, magnesium chloride ( _MgCl₂ ), calcium lactobionate (CaLact), ammonium sulfate (( _NH₄ ) _₂SO₄ ), and calcium sulfate dihydrate ( _CaSO₄ ). Plasticizer: Glycerol was used as a plasticizer. _AS -104 (6%), a low molecular weight (14-30 kDa) compound, was used as the primary membrane-forming component.

Prepare 20 mL of a 1 M salt stock solution based on Table 7. Weigh the appropriate amount of solid salt and dissolve it in 20 mL of deionized water. Once dissolved, stir using a stir plate for 10 minutes. Store the salt solution in the refrigerator. Due to solubility limitations, reduce the calcium lactobionate concentration to 0.5 M.

Stir the prepared saline solution at room temperature for 10 minutes. Keep the saline solution in the refrigerator until use.

Weigh 15 g of AS-104 silk solution and mix it with 0.3 g of glycerol. Place the AS-104 + glycerol mixture on a stir plate and stir for 30 minutes. Then, add 75, 150, 375, and 750 μL of the salt stock solution to the AS-104 + glycerol mixture, giving salt concentrations of 5, 10, 25, and 50 mM. Continue stirring the mixture for 1 hour. Keep the stir bar rpm low enough to avoid foaming. After stirring for 1 hour, transfer the mixture to a vacuum and degas for 1 hour.

Thoroughly clean a silicone mold (3-inch diameter), pre-moisten it with deionized water, and then cast approximately 10 g of the prepared mixture onto the silicone mold. Record the mass of the silicone mold alone and the silicone mold + liquid mixture. Ensure that the liquid spreads across and covers the entire bottom of the silicone mold. Place the silicone mold in an incubator at 35°C and 40% relative humidity. Allow the mixture to dry for 12 hours. Example 6: Test Method

Instron tensile test: After 12 hours, bend the edges of the silicone mold and peel off the film. Trim the edges of the film and keep the middle part of the film. Cut three test areas of 15 mm x 45 mm. Measure and record the film thickness before tensile testing. Mark 10 mm on both ends of the cut film. Use abrasive paper to tap 10 mm and place the sample on the Instron tester with a strain rate of 5 mm/min. Shore A hardness and Veslic test of the film are performed. Example 7: Results Overview Example 8: Combination Formulation Example 9: Water Vapor Transmission Test (WVT) of Leather Test Method:

ASTM Standard E96/E96M-21, “Standard Test Methods for Gravimetric Determination of Water Vapor Transmission Rate of Materials,” (revised edition), ASTM International, West Conshohocken, PA, 2016, astm.org. The purpose of these tests is to obtain reliable values of the water vapor transmission rate through materials, expressed in suitable units, using simple apparatus. These test methods cover the determination of the water vapor transmission rate (WVTR) of materials such as, but not limited to, paper, plastic film, other sheeting, coatings, foam, fiberboard, gypsum and plaster products, wood products, and plastics. Test Information: • Procedure B (Water Method) used • Test temperature: 23.00°C • Relative humidity: 50.00% • The front of the fabric was exposed to water and the back of the fabric was exposed to air • Test equipment: TEXTEST FX 3180 Cupmaster, equipped with an aluminum cup, sealed with NBR and Teflon gaskets, and a screw-on clamping ring • Higher MVTR values indicate more moisture has passed through the material Results:

Water Vapor Transmission Test of Leather with Coating System #1: The results of this test are shown in Figure 7A and Table 12a below.

Water Vapor Penetration Test on Crust Leather (Uncoated) #1: The results of this test are shown in FIG. 7B and Table 12b below.

Water Vapor Transmission Test on Leather with Coating System #2: The results of this test are shown in FIG. 8A and Table 13a below.

Water Vapor Penetration Test on Crust Leather (Uncoated) #2: The results of this test are shown in FIG. 8B and Table 12b below.

Water Vapor Transmission Test of Leather with Coating System #3: The results of this test are shown in FIG. 9A and Table 13a below.

Water Vapor Penetration Test on Crust Leather (Uncoated) #3: The results of this test are shown in FIG. 9B and Table 13b below.

These results indicate that the coated leather allows water vapor to pass through. This demonstrates the breathability of the leather coating while still providing waterproofing. Water droplets cannot pass through, but water vapor can pass through the sample. Example 10: Oil Repellency Testing of Leather

Test method: AATCC TM118-2020, Oil repellency test method: Hydrogen resistance test

Test Information: • Sample size: 8" x 8" • Scale ranges from 0 to 8, with 8 being the most repellent surface. • Allow sample to reach moisture equilibrium; Test conditions: 21°C (±2°C) and 65%RH (±5%RH)

The results shown in Tables 14a-14b indicate that the coated leather, rated 6 out of 8, exhibited significantly better oil repellency than the uncoated leather, rated 0 out of 8. Example 11: Leather Stain Removal Test

Stain Removal Test Method: Oil Removal - AATCC TM130-2018t - Revised Test Information: • Revised = Colorants Used: o French's® 100% Natural Classic Yellow® Mustard o 100% Natural Hunt's® Tomato Ketchup® o Distilled Water o Wine Cube® California Merlot o Topsoil - 25% Topsoil o Mazola Corn Oil o Stars and Stripes Cola o French's® Seasoning Sauce o Artificial Urine • Rating 5 indicates excellent stain removal; Rating 1 indicates very poor stain removal • Allow specimens to reach moisture equilibrium; Test Conditions: 21°C (± 2°C) and 65% RH (± 5% RH) • AATCC Stain Removal Replica 2013 Version was used for grading • Modified handwashing procedure using a soft cloth with a 1% Tide® detergent solution and • 105℉ water • Rinse with a soft cloth and 80℉ water • Air dry Test results :

As shown in Tables 15a and 15b above, the coated leather received all 5/5 ratings, demonstrating excellent stain removal. The uncoated leather did not receive any 5.0 ratings. Example 12: Colorfastness to Light: Xenon Arc Test

Test method : AATCC TM16.3-2020, Test method for color fastness to light: Xenon arc - Option 3

Test Information: • Option 3 - Xenon Arc Lamp, Continuous Light, Black Panel Option • One side of the material is exposed. The test sample is compared to the original, unexposed sample and backed. • Change in shade of the masked portion compared to the original: 5.0 • If the result is not 5.0, the textile is affected by factors other than light, such as heat or reactive gases in the atmosphere. The exact cause is unknown. • QSun Xe-2-HSE xenon test chamber • Horizontal drum specimen rack; 45 × 330 mm dual-panel rack, panel capacity = 15.5 • AATCC EP1-2020, Grayscale Procedure for Evaluation of Color Change • Rating performed under illuminant D65 "Daylight", geometry option C, Gretag Macbeth SpectraLight III • Rating 5 indicates negligible or no color change; Rating 1 indicates significant color change • See Table IV - Reporting Form • Allow specimens to reach moisture equilibrium; rating conditions: 21°C (±2°C) and 65%RH (±5%RH)

Test results: Example 13: Perspiration color fastness test

Test method : Color fastness to perspiration test method - AATCC TM15-2013e

Test Information: • No. 10 multifiber test fabric • Acidic sweat = pH 4.3±0.2 • Alkaline sweat = pH 8.0 • AATCC EP2-2018, Gray Scale for evaluation of staining • AATCC EP1-2018, Gray Scale for evaluation of color change • Rating performed under illuminant D65 "daylight", geometry option C, Gretag Macbeth SpectraLight III • Rating 5 indicates negligible or no staining and negligible or no color change • Rating 1 indicates severe staining and extensive color change • Allow specimens to reach moisture equilibrium; grading conditions: 21°C (±2°C) and 65%RH (±5%RH) • Note: Specimen size increased so cut edges of specimens do not contact edges of fiber strips

Test results: Example 14: Stability of polyurethane and filament systems

Polyurethane/silk systems with medium molecular weight filaments can form gels. The addition of filaments and the type of filaments allow the final product to be adjusted. Example 15: Fourier Transform Infrared (FTIR) Imaging-Heated ATR Imaging

For this FTIR analysis, a JASCO heated ATR-diamond crystal was used. Two liquid silk solution samples (60 mg/mL) were tested.

Instrument Information: Measurement Mode : Attenuated Total Reflectance (ATR). Target: Wide-field ATR microscope "ATR-5000-WG" was used . This instrument performs 1600 × 1600 μm imaging measurements using a single contact point. It is suitable for surface analysis of soft samples such as foreign matter, rubber, and biological samples.

The FTIR results for the plain leather can be seen in Figure 11. The FTIR results for the coated leather can be seen in Figure 13. The FTIR results for the top-coated leather can be seen in Figure 14. Example 16: Fourier Transform Infrared (FTIR) Imaging - Macro ATR Imaging

ATR measurements were performed on coated rubber samples using either a single-element MCT sensor or a 32×32 FPA sensor. Imaging with the FPA sensor and a macro ATR accessory helped resolve the coating distribution details. The results are shown in Figures 15A to 15C. Example 17: Biodecorative Coating System Validation Study

The goal of this study was to document the performance results of the coating system described in this paper and compare them with user requirements. Stain resistance test

A summary of the stain resistance test results for leather treated with the coating system described herein can be found in Table 20 below and in Figures 16A to 16H. Industrial test results

The samples used can be found in Table 21 below and in Figures 17A-17C. The results of this test are summarized in Table 21 below. Wet color fastness friction test / wet Veslic test

The conditions and results of this test are summarized below in Table 22 and depicted in Figures 18A-18I. Barre stretch test

The conditions and results of this test are summarized below in Table 23 and Figures 19A-19D.

The results show that the substrate and coating system passed the Barre flex test up to 20,000 cycles. The substrate and coating system passed the Barre flex test up to 1,000 cycles, up to 5,000 cycles, up to 10,000 cycles, up to 15,000 cycles, and up to 20,000 cycles. The coating system did not separate from the substrate. Tape test

The conditions and results of this test are summarized in Table 24 below. Conclusions • The L1 system has passed the technical user requirements and is eligible for Phase 2 approval. • According to the technical data sheet and safety data sheet provided by the supplier, L5267 is unstable when frozen. Implement temperature-controlled storage and transportation to ensure quality.

Based on the test results described in this article, the coating system of Example 18 is considered validated. Example 18: Tape Test for Basecoat Testing

The following coating formulation was used for this tape test.

Based on the results shown in Tables 26 and 27, there was no delamination. This demonstrates the effectiveness of the coating system described in Table 25.

The samples were then cut in half and ground for three hours, after which the adhesive test was repeated. Grinding involved treating the samples in a dryer for three hours using a mixture of a scrap leather sheet and a wool board as a pressure load. The updated adhesive coating test summary results are shown below in Tables 28 and 29, as well as Figures 21 and 22A-22I.

Based on the results in Tables 28 and 29 and Figures 21 and 22A-22I, the coating system was proven to be effective even after the grinding process.

Samples were also tested after CFR application, before and after a ground base coat. Two HA1 samples were tested by the tape test, one unground before the top coat was applied, and the other ground before the top coat was applied. The results can be seen in Table 30 below and in Figures 24A-24B. Example 19: Leather coating formulation for coating systems

The following formulations summarized in Table 31 are non-limiting exemplary coating recipes according to the coating systems of the present disclosure.

Table 33 provides a brief description of the ingredients included in the exemplary formulation of Table 31 and used throughout this specification. Example 20: Leather base coating

A combination of hydrolyzed silk, gelatin and elastin with plasticizers and a transglutaminase crosslinker to deliver a completely bio-based coating to be applied to leather during the finishing process.

Basecoats are commonly used in the leather manufacturing industry as a pigment delivery method and to cover crust leather repairs. These products are used to modify the leather surface to create favorable tactile and optical properties. During the finishing process, they are combined with a topcoat to produce a color-fast leather product with attractive tactile properties. However, currently available basecoat formulations are (i) derived from petrochemical resources, leading to sustainability issues, and (ii) are non-biodegradable, resulting in problems with processing waste disposal and the disposal of finished leather products.

In some embodiments, the present disclosure provides a product that delivers an optically uniform, stretchable, and elastic coating onto a leather surface and anchors the top coating to the leather. Description of Film Casting 1. Take 40 grams of the prepared base coating solution and pour it into a silicone mold. 2. Place the filled silicone mold in a convection oven at 140°C for 12 hours. 3. Remove the mold from the oven and allow the film to equilibrate for 1 hour. 4. Remove the film from the silicone mold. Solution Preparation ( Crust Leather Adhesive Coating ) 1. Add 414 grams of water to a container. 2. Add 85 grams of low molecular weight silk solution to the solution and stir for 5 minutes. 3. Add 1.25 g of Melios 09s11 crosslinker to the solution and stir for 5 minutes. Solution Preparation ( Base Adhesive Coating ) 1. Add 414 g of water to a container. 2. Add 85 g of medium molecular weight silk solution to the solution and stir for 5 minutes. 3. Add 1.25 g of Melios 09s11 crosslinker to the solution and stir for 5 minutes. Solution Preparation ( Base Coating ) 1. Add 1 kg of water to a container and heat to 60°C. 2. Add 2.5 g of gelatin and 5 g of glycerin to the container and stir for 30 minutes. 3. Remove the solution from the heat source. 4. Add 10 grams of elastin to the solution and stir for 5 minutes. 5. Add 10 grams of transglutaminase/maltodextrin powder and 170 grams of medium molecular weight silk solution to the solution and stir for 5 minutes. 6. Adjust the pH of the solution to 10 with ammonia. 7. Add 20 grams of TP Black E pigment from First Source Worldwide. Solution Preparation ( Top Coat ) 1. Add 500 mL of 100% ethanol to a container. 2. Add 8.3 grams of triethyl citrate and 13.5 grams of prisorine (isostearic acid) to the solution and stir for 5 minutes. 3. Slowly, to prevent clumping, add 12.5 grams of ethyl cellulose to the solution and stir overnight (at least 12 hours). Be sure to stir in a covered container to prevent ethanol evaporation and ethylcellulose concentration. Spraying on Fabric 1. On an unfinished leather surface, apply a coat of crust adhesive using a 1.3 mm spray nozzle in a 45° pyramidal pattern at 35-40 psi, adding 1 gr/ ^ft² of material. 2. Remove the sample and dry it in an oven at 130°C for 30-60 seconds (until dry). 3. Next, spray the base coat onto the leather again using a 1.3 mm spray nozzle in a 45° pyramidal pattern at 35-40 psi, adding 1 gr/ ^ft² of material. 4. Remove the sample and dry it in an oven at 130°C for 30-60 seconds (until dry). 5. Iron the sample using a roller iron at 98°C, 50 kg/f pressure and a speed of 6 m/min. 6. Apply two more coats of the base coat formulation using steps 3 and 4. 7. Finally, spray the top coat formulation onto the leather using a 1.3 mm nozzle in a 45° pyramidal pattern and 45-50 psi, adding 1 gr/ ^ft2 of material. 8. Remove the sample and dry it in an oven at 130°C for 30-60 seconds (until dry). 9. Repeat steps 7-8 two more times. 10. Iron the sample using a roller iron at 98°C, 50 kg/f pressure and a speed of 5 m/min. Performance Test Method Film Test

All films undergo internal qualitative testing based on bendability, stretch, elasticity, and water drop resistance. Each test is performed on a scale of 1-4, with 1 being the worst and 4 being the best. - For bendability, the film is creased in two directions. If the film cannot bend, a score of 1 is assigned, and if the film can be creased without leaving a mark, a score of 4 is assigned. - For stretch, the film is pulled in opposite directions. If it breaks without any stretching, a score of 1 is assigned, and if it can stretch to more than twice its size, a score of 4 is assigned. - For elasticity, the film is stretched. If it cannot be stretched any distance without permanent deformation, a score of 1 is assigned, and if it can be stretched to twice its length and still recover its shape, a score of 4 is assigned. - For water drop resistance, a drop of water was applied to the surface using an eye dropper. If the film dissolved immediately, a score of 1 was awarded, and if no change occurred after the water was applied and evaporated, a score of 4 was awarded. Leather Test

All coated leather samples were tested using the Veslic test to ensure color fastness to rubbing and the Bally flex test to ensure adhesion between all layers and prevent cracking. The Veslic test followed the ISO 11640 test procedure and the Bally flex test followed the ISO 5402 test procedure. Test results Film test results

All membrane test results can be found in Table 34. Based on the membrane test data, the silk/elastin/gelatin formulation cross-linked by transglutaminase produced the best membranes. In all formulations, activated silk (AS-105) and gelatin were present to provide the membrane structure and bulk. Glycerol was also added to the membrane as a plasticizer to increase flexibility. Initially, Etocas 200 (highly ethoxylated castor oil) was added.

As seen in Experiments 1-3, the films were made soft, however, flexibility was sometimes below standard, and the films had little stretch and elasticity, as well as poor water resistance. To increase water resistance and stretch, Span 120 (sorbitan isostearate) was used instead of Etocas 200. This increased water resistance, however, like Etocas 200, it lacked stretch and elasticity, as seen in Experiments 4-5.

Elastin was used to increase stretchability, and transglutaminase was tested to improve elasticity. As seen in experiments 7-18, stretchability and elasticity were greatly improved when elastin and transglutaminase were added. To find the optimal concentration, standard curves were made for each component, as seen in experiments 19-36, and the optimal concentration was retained.

The ratio was 0.25:1:1 gelatin to transglutaminase to elastin. After determining the optimal ratio, the optimal concentration of total non-silk solids in the solution was investigated, resulting in an optimal concentration of 2.75% additive. Leather Coating

For each leather coating experiment, four layers were applied. First, an adhesive layer consisting of a low-molecular-weight activated silk (AS-104) and a Melios-09s11 crosslinker was applied to the leather's outer skin. Next, a base coat was applied at an application rate of 3 g/ft². An adhesive layer consisting of a medium-molecular-weight activated silk (AS-105) and Melios-09s11 was then applied between the base coat and the top coat. Finally, the top coat was sprayed on. Veslic test results

All Veslic test results can be found in Table 35. For the two samples without a basecoat and with Etocas 200 as the primary additive, if the topcoat contained isostearic acid, it would be rubbed off, causing the pigment to leach onto the scrubbing pad, e.g.

As can be seen in Experiments 1-3. However, if Span 120 is used in the top coat, it will last up to 300 cycles, as can be seen in Experiments 4-5. If Span 120 is used in the base coat together with a crosslinker, the top coat will prevent the pigment from leaching onto the scrubbing pad. However, the leather surface will deform after rubbing (except when using Rodalink 3315 crosslinker), and the use of petrochemically derived

The crosslinker prevents this formulation from being 100% biobased. However, when elastin is used with transglutaminase, there is no pigment bleeding and the surface remains unchanged, as seen in Experiment 12. The use of elastin and transglutaminase also makes the formulation 100% biobased. Barre test results

All Barre Twist test results can be found in Table 36. Using Etocas 200 in the basecoat resulted in the sample experiencing complete detachment of the topcoat from the basecoat at 8,000 cycles, as seen in Experiments 1-2. In addition, by removing Etocas 200 from the basecoat, the sample did not experience complete detachment, however, it still experienced slight detachment, as seen in Experiments 3-4. Even though

Experiments containing Span 120 in the basecoat still experienced slight desorption, as seen in experiments 5-6. However, samples containing elastin and transglutaminase in the basecoat did not experience any desorption up to 8,000 cycles. Example 21: Basecoat and Topcoat Components for a Coating System

The following are examples of amounts of various products that may be included in the topcoat and basecoat according to the coating systems described herein. Case 22: BIOPUR 3015 Adhesive Study

Evaluate the minimum feasible amount of BIOPUR 3015 required to pass the adhesion test.

As can be seen in Table 38, the cutoff value for passing the Scotch Tape test was approximately 0.5% BIOPUR 3015.

To further assess the change in performance, water resistance was measured by adding a drop of water to the surface and observing whether it absorbed into the leather over a 5 minute period.

This was then studied by adding different levels of 10% BIOPUR 3015.

To better understand the effect of activated silk on topcoat adhesion, adhesive layers were prepared with BIOPUR 3015 and the minimum amount of silk required to achieve 80% biobased content. Example 23: Challenges of matting agent system and designing matting agent with L1 system

Loading capacity of 2.5% ethyl cellulose: (i) low solids loading in the topcoat typically limits total matting agent to <2%; (ii) silica is required for adequate matting at low concentrations (relative to mineral, clay, or cellulose-based matting agents); and (iii) poor relative polish resistance in dispersed silica coatings.

Matt depth and polish resistance: (i) for the same Δgloss, darker matte coatings show a more visible polish; (ii) reducing the matte level to make the coating more easily polished produces an overly glossy finish; and (iii) the best silica matting agent (Acematte® 3300) shows a visible polish at concentrations > 0.40%.

Performance test: Trade-off between Barre twist and silica content. System design

Design strategy: 2-part matting agent.

Minor Component: Silica. Silica at a concentration of 0.25-0.40% provides most of the gloss reduction at a concentration low enough to avoid polishing. In final form, 3.5-5% of an interchangeable plasticizer solvent is incorporated.

Main component: polymer host. Polymer (PU, Decosphaera®) at a concentration of 0.50-1.00% provides additional gloss reduction while being fully polish resistant. The polymer also acts as a host for silica, preventing polishing. Formulation A

The original Formulation A is presented in Table 39 , and the modified Formulation A (stable formulation) is presented in Table 40 . Example 24: Formulation A: L1 Matting Agent. 2- Part Matting Agent System

A schematic diagram of the preparation of a two-part matting agent system is presented in Figure 50. Matting Agent A ● Silica matting agent: Acematt® 3300 - 5.0%. ● Stabilizer: Aerosil® R972 - 0.5%. ● Solvent (plasticizer): Triacetin - 94.5%. ● The silica matting agent, stabilizer, and solvent are stirred to provide a silica solution ( i.e. , solution (a) or matting agent A). Matting Agent B ● PU matting agent/host: Decosphaera® - 40.0%. ● Stabilizer: Solagum ^™ Tara - 0.2%. ● Solvent: Water - 58.8%. ● Stir the PU matting agent/host, stabilizer, and solvent to provide a PU dispersion ( i.e. , solution (b) or matting agent B). Topcoat Solution ● Dilute 5% ethyl cellulose in 50% MP into 50% MP to provide a topcoat solution ( i.e. , solution (c)). Matting Topcoat ● Add Matting Agent A (5.00-8.00%) and Matting Agent B (1.25-2.50%) to the topcoat solution (90-93%) to provide the final formulation, the matting topcoat. Exemplary matting topcoat formulations are shown in Table 47 . Example 25: Plasticizer Testing Evaluation Step 1: Initial screening of cast films with an ethylcellulose topcoat for flexibility, uniformity, and migration/separation. Step 2: Candidate plasticizers were tested on one leather type and evaluated for visual appearance (cracking, discoloration, lines, white spots), color fastness to rubbing (CFR), adhesion (tape test), and water droplet resistance (through the finish) after sanding. Step 3: Candidates from the shortlist were further tested on three additional leather types. Testing was performed before and after sanding for visual appearance, barre deflection, gloss, CFR, tape test, and water droplet resistance. Hand feel was also evaluated.

Candidates that pass step 3 are sent to generate products for customer feedback. Step 1 - Initial Screening

Candidate solvents are presented in Table 48 .

Generally speaking, the glycerol and hydroxyl groups (polyols) show better performance than other groups. Step 2 - Next

Selected PLs are shown in Table 49 , and the trade names and chemical names are shown in Table 50 . Step 3 - Leather Type Assessment

The leather type evaluations before and after grinding are presented in Table 51 and Table 52 , respectively. Figures 51A , 51B , and 51C show exemplary gray , brown, and black leathers with CAP -7, MHG, and DPGDB, respectively. Example 26 : Top coating using nozzles of different diameters. Setting the spray material content

The pressure is set to 4 bar. Procedure for producing the top coat using nozzles of different diameters

Use 2.5%, 5%, and 7% ethyl cellulose. Use a 6 x 6 inch brown sheet with a standard base coat of L5267. Apply four coats and measure the mass before and after each coat. Calculate the amount of material per square foot. Viscosity of ethyl cellulose

Using an LV-02 (62) speed of 1.5 rpm, the viscosities of ethyl cellulose at three different concentrations in methoxypropanol were: (i) 2.5% ethyl cellulose, 120 cps; (ii) 5.0% ethyl cellulose, 1480 cps; and (iii) 7.0% ethyl cellulose, 8520 cps.

The weight of the coated samples and the diameter of the nozzle are presented in Figure 52. Images of samples with ground and unground crust leather at different ethyl cellulose concentrations in methyl propanol are presented in Figures 53-53F . Example 27: Matting Test of Formulation A.

Figure 54 shows the test results of the extinction test of Formulation A. The SADESA laboratory test standards (section: potency) are presented in Table 54 .

Additional tests for WP products are presented in Table 55 . Example 28: Matting agent test of formulation A.

Formulation A screening on L5267 is presented in Tables 58 and 59 .

Formulation A screens using all additives are presented in Tables 60 and 61. As shown in the tables, there was some loss of gloss after ironing, as expected using a polished panel, but after grinding, the gloss units were similar to before ironing, and the color did not change significantly.

Figures 55A- 55N show images of the Euroleather (top) and Fracopel (bottom) before and after ironing.

Figures 56A- 56N show images of Fragopel and Euroleather after grinding (top) and before grinding (bottom). Ceral 63/N (a palm wax-based additive) was found to significantly crack and delaminate the top coating after 8 hours of grinding.

Tables 62 and 63 show the screening of Formulations A, B, C, and D on L5267.

Figures 57A- 57L show the Euroleather (left) and Fracopel (right) using formulations ABCD before ironing, after ironing, and after grinding. All samples before ironing showed some spots, in the following order: formulations C, B, A, D. After ironing, all spots disappeared. After grinding, all samples fractured in the following order: formulations D, C, A, B.

Figures 58A and 58B show the vulcanization test.

Tables 64 and 65 show the test results for Formulation 061.

Tables 66 and 67 show the test results of Formulation 061 with Fraction 24.

Tables 68 and 69 show the test results of Formulation 072 with Fraction 24. General Observations for 24 -part Formulation 072 : ● 072-1: Part AUX-SAS-071-1 required mixing before preparing the application formulation. Viscosity: 119.1 cp. Appearance was inconsistent and speckled on both brown and black Fracopel. ● 072-2: The application solution was not filtered directly; it required manual force through the filter using a stirrer. Viscosity: 136.5 cp. ● 072-3: Viscosity: 104.7.

Figures 59A- 59C show the paint resistance of leather treated after spraying and before ironing. Example 29: Matting Agent Summary Data

Table 70 shows the matting agent summary data.

Table 71 shows the experimental overview.

Selected formulations are presented in Table 73 .

Table 74 shows the matting agents and waxes evaluated.

Table 75 shows the reference data.

Table 76 shows a list of matting agents.

Table 77 shows the polish resistance list.

Table 78 shows exemplary properties of samples of Formulations A, B, C, and D.

Table 79 shows exemplary properties of samples of Formulation 052. Example 30: LUMOS II characterization of rubber samples.

Rubber samples were characterized using a LUMOS II stand-alone FT-IR microscope.

ATR measurements are performed on coated rubber samples using a single-element MCT sensor or a 32×32 FPA sensor. Imaging with the FPA sensor and the giant ATR accessory helps resolve the coating distribution details.

Figure 60 shows the IR spectrum of a sample obtained using an LN-MCT detector. Figure 61 shows a macroscopic ATR image of a sample with an adhesive undercoat. Figure 62 shows a macroscopic ATR image of a sample with a topcoat. Example 31: SEM image of a sample tested using BSE (SE2).

Figure 63 shows a cross section of uncoated leather. Surface unevenness is visible.

Figure 64 shows a cross section of leather coated with a primer. The primer was sprayed with a 4 g/sqft primer: (1) 2 passes of 1 g/sqft; (2) ironed at 90°C with a pressure of 50 kg/cm and a drum speed of 6 m/min; and (3) 2 passes of 1 g/sqft. A smoother surface than the uncoated leather can be seen. The primer contains 0.4% silver-marked silk. Due to the low silk concentration, the silk distribution in the coating is low. The coating thickness is 3 to 5 microns.

Figure 65 shows a coated leather with the L1 system. The base coat was sprayed with a 4 g/sqft base coat and contained a 0.4% silver marker thread: (1) 2 passes of 1 g/sqft; (2) ironed at 90°C with a pressure of 50 kg/cm and a drum speed of 6 m/min; and (3) 2 passes of 1 g/sqft. The top coat was sprayed with 6 g/sqft: (1) 1 pass of 6 g/sqft, and (2) ironed at 90°C with a pressure of 50 kg/cm and a drum speed of 6 m/min. The thickness of the top coat was 2 to 3 microns. After treatment, the top coat is integrated with a base coat that has a water-repellent surface.

Figure 66 shows a closer look at the silver markers in the L1 system. The basecoat/topcoat composite is indicated by the silver markers throughout the coating.

Figure 67 shows a schematic diagram of the resulting layers. The heat and pressure during processing create a composite layer between the topcoat and basecoat, with the heavier concentration of basecoat closer to the bottom surface and the topcoat concentration higher at the surface. The topcoat is completely intact with respect to water repellency, as confirmed by wet veslic testing. Example 32: Scale-up Method Example 33: Modified Peptide

A novel method is disclosed for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides. These two novel compositions are referred to as low-sled and mid-sled/modified polypeptide compositions.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, using carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperatures to remove excess water, preserving the polypeptide composition. The silk is then dissolved in a high concentration of lithium salt at two different temperatures and times to produce different compositions of medium and low-density silk. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The low- and medium-sled silk/modified peptide compositions contain populations of silk/modified peptides with unique properties. The low-sled silk/modified peptide composition did not self-assemble at 5 mg/mL. The low-sled silk/modified peptide composition contained two major populations of silk/modified peptides: one population (AS22), which did not self-assemble under conditions that promote self-assembly at 5 mg/mL and was enriched in negatively charged amino acids; and a second population (AS12), which rapidly self-assembled at 5 mg/mL and was depleted of negatively charged amino acids.

When AS12 and AS22 are combined at a 50% ratio, the average molecular weight of the mixture becomes the same as that of the low-sludge/modified peptide composition. Therefore, the low-sludge/modified peptide composition is composed of 50% AS12 and 50% AS22 peptide/modified peptide compositions.

The mid-sled filament/modified polypeptide composition contained two major populations of filament-modified polypeptides; one population (AS11) self-assembled more slowly than the mid-sled filament/modified polypeptide under conditions promoting self-assembly at 5 mg/mL and was enriched in negatively charged amino acids; and a second population of polypeptides (AS1) self-assembled faster than the mid-sled filament/modified polypeptide under conditions promoting self-assembly at 5 mg/mL and was depleted in negatively charged amino acids.

When AS11 and AS11 are combined at a 50% ratio, the average molecular weight of the mixture becomes the same as that of the medium-strength/modified peptide composition. Therefore, the low-strength/modified peptide composition is composed of 50% AS1 and 50% AS11 peptide compositions.

Both the low-sled and medium-sled/modified peptide compositions contained modified peptides as determined by mass spectrometry.

The low- and medium-slaughter silk/modified polypeptide compositions described in this disclosure have never been produced before. These silk-derived and modified polypeptides exhibit a wide range of behaviors upon isolation, from extreme self-assembly to solubility and stability over time in various buffers, as well as a variety of average molecular weights and polydispersities. These novel silk-derived polypeptide compositions contain unique modified amino acid sequences resulting from a unique pharmacopoeial processing method and scale-up. Strict control over temperature, concentration, salt concentration, physical agitation, and purification allows us to tailor unique peptide compositions from native/modified complex species at each step in the process to specific performance criteria. Some constitutive polypeptide compositions exhibit biological activity and are useful as treatment candidates.

HETs are versatile materials with applications ranging from the development of implantable medical devices to the development of pharmaceutically valuable soluble peptide formulations. A major challenge with peptides in solution is their tendency to self-assemble and aggregate, making controlling their solubility extremely difficult. Furthermore, the kinetics of gel/film formation cannot be controlled predictably. This novel amide/modified peptide composition contains a population of peptides, allowing for control of their properties and the development of products with predictable and desired properties.

The disclosed compositions contain a range of polypeptides with diverse properties. Interfacial activity is primarily characterized based on molecular weight and polydispersity, and mixtures of interfacially active/modified polypeptides are either uncharacterized or produced. As disclosed herein, a unique large-scale process is used to produce combinations of modified/modified polypeptides.

Low/medium sled silk begins its degumming process with sodium carbonate at specific silk mass, sodium carbonate, and water ratios. Multiple wash cycles at different temperatures—100°C and 60°C—as well as agitation, are crucial in producing specific natural/modified compositions. The silk is then dried to remove water while maintaining the desired composition. Next, the silk is dissolved in highly concentrated lithium bromide at 103°C and 125°C for 1 hour and 6 hours, respectively. The time and temperature allow for fine-tuning the degree of post-translational modification, resulting in unique peptide compositions. The silk is then purified to remove the lithium bromide and, if necessary, concentrated. For the downstream characterization of the isolated various silk/modified peptide compositions, analytical techniques, biochemical/biophysical techniques, and cell biology approaches were employed.

To characterize/isolate novel low- and medium-stranded/modified peptide compositions, a combination of ion exchange chromatography fractionation, analytical methods, and biochemical anatomy was used to characterize their properties. Generation of low- and medium-stranded / modified peptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the silk glue and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 103°C for 1 hour (for medium silk) and at 125°C for 6 hours (for low silk). This dissolution step controls not only the molecular weight but also the polypeptide modifications that produce the natural/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the natural/modified silk composite and pure water. The temperature, time, concentration, stirring and shear of each unit operation are strictly controlled. Separation of low and medium sled / modified peptide compositions Separation of AS22 sled / modified peptide compositions

To isolate AS22, the low-sludge filament/modified peptide complex was fractionated using HiTrap Q Sepharose anion exchanger (Figures 68 and 69). Tris was added to the filament preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The filament was centrifuged and then loaded onto a HiTrap Q Sepharose column to remove any pre-formed aggregates. The low-sludge filament preparation solution has a characteristic yellow hue. The effluent from the HiTrap Q Sepharose column is clear. AS22 was eluted with 1 M NaCl and has a strong yellow hue.

When the AS22 formulation was analyzed by HPLC using an analytical SEC column (see Materials and Methods), it had an average molecular weight (MW) of approximately 35 kDa and a polydispersity (PDI) of 2.3 ( Figures 70 and 71 ). This molecular weight and polydispersity were unique and different from those of the low-stranded/modified polypeptide composition (MW approximately 19.5 kDa and PDI 2.2) and the medium-stranded/modified polypeptide composition (MW approximately 38 kDa and PDI 2.4) ( Figures 70 and 71 ).

When AS22 and low-sled silk were analyzed in isoelectric focusing polyacrylamide gels, 22M silk peptides were found to have pIs of 3-6 with some species having a pI of approximately 9.6, while low-sled silk also contained peptides spanning the entire range of pIs from 3 to 9.6 (Figure 72). Separation of AS12 silk / modified peptide composition components of the low-sled silk / modified peptide composition

To develop AS12, a combination of ion exchange chromatography (IEX) fractionation was used to purify the preparation, and analytical methods and biochemical dissection were used to characterize its properties.

To isolate AS12, a low-slugging silk preparation was fractionated using HiTrap Q Sepharose anion exchanger (Figures 68, 69). Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl pH = 8.0. The silk was centrifuged and then loaded onto a HiTrap Q Sepharose column to remove any pre-formed aggregates. The effluent from the HiTrap Q Sepharose column was collected and named AS12. AS12 is colorless. The AS12 silk preparation is composed of short silk / negatively charged, modified polypeptides.

When AS12 was analyzed by analytical size exclusion chromatography, it had an average molecular weight (MW) of approximately 12 kDa and a polydispersity (PDI) of 1.7 (Figures 70 and 71). This molecular weight and polydispersity were unique and distinct from the low-sled/modified peptide composition (MW approximately 19.5 kDa and PDI 2.2) and the medium-sled/modified peptide composition (MW approximately 38 kDa and PDI 2.4) (Figures 70 and 71). When AS12 and the low-sled/modified peptide composition were analyzed in isoelectric focusing polyacrylamide gels, the AS12 filament peptides were found to have a pI of approximately 9-10, while the low-sled composition also contained peptides spanning a pI range of 3 to 9.6 (Figure 72), with the majority concentrated around a pI of 3-5.5. Separation of AS1 filament / modified polypeptide components from the filament / modified polypeptide composition

To isolate AS1, the low-sled silk preparation was fractionated using HiTrap Q Sepharose anion exchanger (Figures 68 and 69). Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl pH=8.0. The silk was centrifuged and then loaded onto a HiTrap Q Sepharose column to remove any pre-formed aggregates. The effluent from the HiTrap Q Sepharose column was collected and named AS1. The MW of AS1 is approximately 28 kDa and the PDI is 1.7-2.1 (Figures 70 and 71). The MW of the mid-sled silk/modified peptide complex is 37 kDa PDI 2.0 (Figures 70 and 71). Separation of the AS11 silk / modified peptide complex component of the mid-sled silk / modified peptide complex

To develop AS11, a combination of ion exchange chromatography (IEX) fractionation was used to purify the preparation, and its properties were characterized using analytical methods and biochemical anatomy. To isolate AS11, a filament/modified peptide combination preparation was fractionated using HiTrap Q Sepharose anion exchanger (Figures 68, 69). Tris was added to the filament preparation to a final concentration of 50 mM Tris-HCl pH = 8.0. The filament was centrifuged and then loaded onto a HiTrap Q Sepharose column to remove any pre-formed aggregates. The effluent from the HiTrap Q Sepharose column was collected and named AS11. The molecular weight (MW) of the AS11 formulation was approximately 53 kDa and the polydispersity (PDI) was 2.8 (Figures 70, 71). This molecular weight and polydispersity are unique and distinct from the medium-stranded/modified peptide compositions (MW, approximately 37 kDa and PDI 2.4) (Figures 70 and 71). Self-assembly of low- and medium-stranded / modified peptide compositions Self-assembly analysis and data derived therefrom.

To investigate the stability of AS1 in solution, a self-assembly assay was performed. AS1 self-assembles very rapidly at 5 mg/mL. The absorbance curve at 550 nm from the self-assembly assay is S-shaped and can be described as a logistic curve. A typical logistic function is: fx = Amax1 + ek(t - t0.5), where _Amax is the maximum density of the formed gel, k is the self-assembly rate factor, _t0.5 is the time point at which 50% of the gel is formed, and e is the exponential equation for the specific curve (see the dashed red line in Figure 73A to better illustrate how these factors are calculated for the self-assembly experiment).

Another parameter introduced to characterize the tendency of filaments to form gels is the self-assembly factor, which is: f _SAF = 1t _0.5 × A _max × 1000

Using experimental data from self-assembly analyses of various newly isolated peptides, these parameters were calculated and used to dissect their properties (Figures 73, 74, and 75). Four parameters, collectively referred to as self-assembly kinetic factors, are the self-assembly rate factor (SARF), _Amax , t0.5, and self-assembly factor (SAF) (Figures 73, 74, and 75). SARF indicates how quickly filaments self-assemble to form a gel after the reaction begins or the gelation core forms; _Amax indicates the density of the formed gel after self-assembly is complete; t0.5 indicates how long it takes for the self-assembly reaction to reach a point where the gel density is Δmax2; and SAF indicates the direction of filament self-assembly (Figures 73, 74, and 75). The AS1 filament / modified peptide combination exhibits the fastest self-assembly kinetics.

As previously described, self-assembly analysis revealed that AS1 self-assembles very rapidly, much faster than the mid-sled filament/modified peptide combination (Figures 73, 74, and 75). The mid-sled filament/modified peptide combination served as a positive control and exhibited rapid self-assembly kinetics (Figures 73, 74, and 75). The AS11 filament / modified peptide combination exhibited the fastest self-assembly kinetics.

As previously described, self-assembly analysis showed that AS11 self-assembled rapidly, but not faster than the mid-sled/modified peptide combination (Figures 73, 74, and 75). The mid-sled/modified peptide combination served as a positive control and exhibited rapid self-assembly kinetics (Figures 73, 74, and 75). The AS12 amide / modified peptide combination is a component of a low-skill amide / modified peptide combination that promotes self-assembly.

As previously described, self-assembly analysis showed that AS12 self-assembled rapidly, but not faster than the mid-sled filament/modified peptide combination (Figures 73, 74, and 75). The mid-sled filament/modified peptide combination served as a positive control and exhibited rapid self-assembly kinetics (Figures 73, 74, and 75). The AS22 filament / modified peptide combination was very stable in aqueous solution and did not self-assemble.

As previously mentioned, self-assembly analysis revealed that AS22 exhibited remarkable stability and did not self-assemble even under conditions that promote spherical self-assembly. A medium-sled filament was used as a positive control and exhibited rapid self-assembly kinetics (Figures 71, 72, and 73). Combining AS1 , AS11 , AS12 , and AS22 filament / modified polypeptide compositions at varying ratios yielded compositions with unique properties.

To better understand the properties of the filament/modified polypeptide compositions (Figures 73, 74, and 75), a series of mixtures were generated (see Table 1). The resulting compositions exhibited unique combinations of properties in self-assembly assays (Figures 73, 74, and 75). Mixtures of AS1 and AS11 (AS2-10) and AS12 and AS22 (AS13-AS21) all exhibited unique combinations of self-assembly kinetics (Figures 73, 74, and 75). This information can be used to generate fiber mesh/modified polypeptide compositions with specific desired properties. Materials and methods used to generate and characterize AS1-AS28 . Anion exchange fractionation separation.

A low-sludge filter was provided at a concentration of 60 mg/mL. The filter was centrifuged at 20,000 × g for 15 min at 4°C to remove any aggregated material. For fractionation using columns connected to an AKTA Pure 25 µL or packed with Q-Big beads, Q-Sepharose prepacked columns were used. All buffers used were filtered through a 0.45 μm PES filter and degassed by sonication. Load a centrifugal low- or medium-sled filter onto a 5 × 5 mL HiTrap Q HP column and wash with 10 column volumes of 50 mM Tris pH 8.0, 10 column volumes of 50 mM Tris pH 8.0, 1 M NaCl, and a final 10 column volumes of 50 mM Tris pH 8.0. Load 100 mL of the centrifugal low-sled filter onto the column at a flow rate of 5 mL/min. Collect the flow-through. Elute the column with 50 mM Tris pH 8.0 until the absorbance at 280 nm ( _A280 ) reaches 100 AU. Elute the bound protein in a single step with 50 mM Tris pH 8.0, 1 M NaCl, and all fractions with an absorbance ( _A280 ) > 500 AU. 20 mL of the low-sludge (LS) fraction, flow-through (QFT), and pooled elution fractions (QE) used for fractionation were placed in a dialysis bag (Sigma D9652-100FT, dialysis tubing cellulose membrane, average flat width 33 mm, 1.3 inches) and immersed in a 50× volume of 50 mM Tris pH 8.0. The samples were quickly frozen in liquid nitrogen and stored at −20°C until use. Prior to use, the samples were slowly thawed at 4°C or on ice. Analytical / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration was determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation was diluted until the _A280 was between 0.1 and 1. Within this range, absorbance correlated linearly with the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution was calculated after adjusting the dilution for absorbance measurements. Analytical size exclusion chromatography was performed.

Analyses were performed on a 300 mm × 7.8 mm PolySep GFC P-4000 LC column (Phenomenex, part number CHO-9229) connected to an Agilent 1260 Infinity II HPLC system equipped with an Agilent G7162A _RID refractive index detector. The mobile phase used for the analysis was a 0.1 M NaCl, 12.5 mM _Na₂HPO₄ solution, pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass culture medium bottle). A 25 μL sample was loaded onto the column and analyzed at 25°C at a flow rate of 1 mL/min for 20 min. Molecular weights of each sample were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC&LC/MS Systems software, Cirrus SEC data collection, and molecular weight analysis software. Analytical anion exchange chromatography was performed.

For analysis, a Dionex Ultimate 3000 HPLC column with a 7.5 mm ID x 7.5 cm, 10 μm TSKgel DEAE-3SW column was used. Chromatography was performed at 25°C. A 10% trifluoroethanol, 45% acetonitrile/water solution was used as _the raw material. The column was equilibrated with 20 mM _Na₂HPO₄ / _{KH₂PO₄ (} Na₂HPO₄: _Fisher Chemical, _Lot # 188298, PN# S374-500, _KH₂PO₄ : Fisher Chemical, _Lot # 187270, PN# P285-500), pH 6, at 1 mL/min for 10 minutes. 30 μL of sample was loaded onto the column. The bound peptide was eluted using a linear gradient of 500 mM NaCl. All solutions were prepared in LCMS water: Fisher Chemical, lot number 216650, PN number W5-4. Data were analyzed using XCalibur ^™ software. Isoelectric point determination protocol.

To determine the isoelectric point of the new composition, isoelectric focusing gels were used. These separate proteins based on their net charge rather than their molecular weight. For analysis, BIO-RAD standard precast gels were used, EEF standard pI 4.45-9.6. Protein samples from the new composition were mixed with EF sample buffer (ensuring at least 5% v/v glycerol in the final mixture). The mixture was loaded on a Criterion Precast Gel. For electrophoresis, 1× IEF anodic buffer and 1× IEF cathodic buffer were used. The running conditions for electrophoresis were: 100 V constant for 60 minutes, 250 V constant for 60 minutes, and 500 V constant for 30 minutes. After electrophoresis, proteins were fixed on the gel using a solution of 40% v/v methanol (Sigma Aldrich, Methanol ACS Reagent >99.8%, 179337-4L-Pb, Source SHBN0806, Pcode 1003210445) and 10% v/v acetic acid for 30 minutes or overnight at room temperature with rocking. Peptides were analyzed by LC/MS .

Store the samples at 4°C until analysis. For each sample, take an aliquot and mix it with an equal volume of 6 M guanidine hydrochloride (GuHCl) in a new tube. From this mixture, take another aliquot to produce 120-fold and 240-fold additional dilutions for determination of protein concentration using the BCA assay. Using the concentration determined above, dilute the samples to 20 μg/μL with 6 M GuHCl. Transfer an aliquot of 1,000 μg of total protein to a new tube. Add 50 mM dithiothreitol (DTT) to a final concentration of 5 mM and incubate the samples at 60°C for 30 minutes. After a brief equilibration period at room temperature, 100 mM iodoacetamide (EtOAc) was added to a final concentration of 10 mM, and the samples were incubated in the dark at room temperature for 30 minutes. The reaction was quenched by adding 50 mM DTT to a final concentration of 5 mM DTT, followed by a further 30 minutes of incubation at room temperature. Three aliquots corresponding to 100 μg of total protein were dissolved in separate tubes and diluted in PBS to a final concentration of 0.2 M GuHCl. The samples were then treated overnight at room temperature (trypsin) or 37°C (trypsin/Lys-C and Glu-C) with a protease to protein ratio of 1:50 (2 μg of each protease). The protease reaction was quenched by adding TFA to a final concentration of 1% (v/v). The sample was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was transferred to an HPLC autosampler vial for LC-MS analysis. LC column: C18 column (100 μm × 150 mm, 3 μm) Mobile phase A : Water with 0.1% formic acid Mobile phase B : Acetonitrile with 0.1% formic acid Flow rate: 600 nL/min (micropump) and 15 μL/min (load pump) Run time: 60 min Gradient: 7% B (0 to 3.3 min); 7% to 35% B (3.3 to 35 min); 35% to 95% B (35 to 37 min); 95% to 80% B (37 to 39 min); 80% B (39 to 41 min); 80% to 7% B (41 to 44 min); 7% B (44 to 60 min) Injection volume: 4 μL

The full MS acquisition range was 350 to 1600 m/z. The MS method was based on data-dependent acquisition (DDA) of the top 10 ions with an isolation window of 3.0 m/z and a normalized collision energy of 26.

Data were acquired using Thermo Xcalibur™ software. Data analysis was performed using Thermo Proteome Discoverer™ software. To unambiguously assign specific proteins to the identified peptides, at least two unique peptides per protein were required when searching the SwissProt database. Gel staining methods. Silver staining

SDS and EF polyacrylamide gels were stained using the ProteoSilver silver staining kit according to the manufacturer's instructions. Briefly, gels were immersed in a fixative solution (50% v/v ethanol, 10% glacial acetic acid) and washed with water, a sensitizer solution, a silver solution, and a developer solution. Development of the gel was terminated with ProteoSilver stop solution. Self-assembly analysis was performed .

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM _CH₃COONa , pH 5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. A buffer of 50 mM _CH₃COONa , pH 5, and 35% v/v 2-propanol was first prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid the formation of any bubbles. Record the absorbance at 550 nm for 16-24 h (depending on the sample). Export the recorded values to an Excel file for storage and further analysis. Example 34: Preparation and separation of low molecular weight and medium molecular weight fibrous proteins

Fibrin separation requires the separation of serine from the original B. mollissima fibers. This separation is performed in a single stage and facilitated in a solid extraction operation. The unit consists of an atmosphere container enclosing a perforated bubbler that rotates on a horizontal axis. The raw sugars are loosely packed in permeable mesh bags before the raw fibers are loaded into the rotating drum via a sealable access port. This primary blockage prevention minimizes product losses, protects the equipment by preventing the flow strands from tangling with the rotating components in the container, and protects the discharge line from becoming blocked by solids that would otherwise escape the discharge hopper during processing.

The vessel is filled with an extraction solvent comprising an aqueous solution of 0.7% to 0.95% wt. (typically 0.94% wt.) sodium carbonate to partially immerse the perforated tank. This solvent composition has been shown to effectively dissolve and stabilize serine in solution. The argon/solvent ratio is 0.040 kg/kg - 0.070 kg/kg (typically 0.042 kg/kg). The temperature of the extraction solvent is maintained in the range of 94.5°C to 97°C using an electric heater located in the base of the vessel. Maintaining the extraction solvent at an elevated temperature drives the serine solution to form. Second, the high temperature thermally cleaves the fibrous protein chains to reduce the average molecular weight of the protein population. The rotary drum is rotated periodically during the 30-minute isothermal phase of the extraction. This action serves to expose all fiber surfaces to the extraction solvent.

Rinse with concentrated hot water. Fill the container with non-perforated water to partially submerge the perforated bubbler. Maintain the rinse water temperature between 55°C and 65°C for 20 minutes with intermittent bubble rotation, then drain the rinse water to waste. Repeat this two more times.

After rinsing, the drum rotates at high speed to remove water retained in the colon through centrifugal action. The wet swab of fibrous protein, with a moisture content ranging from 15% to 65% by weight (average 46.74%), is then manually removed from the pad and evenly distributed onto a perforated tray. Residual moisture is removed by storage in a dryer maintained at an internal temperature of 55°C to 60°C, until the moisture content of the material is less than 1% of the total weight.

The extraction efficiency was verified by measuring the change in mass of the dried material before and after treatment in the extraction unit. Typically, the amount of serine removed from the colon was 30-36% by weight of the total mass of the original colon. The composition of the original cocoons was characterized by LCMS. The method screened for bile proteins, fibronectin (P25), heavy-chain fibronectin, light-chain fibronectin, serine, and trypsin inhibitors. Using LCMS, the serine concentration in the original colon was determined to be approximately 35.27 wt%. After treatment in the extraction unit, serine was undetectable in the fibronectin. The results indicate that the serine extraction method is effective and that the fraction of serine detected in the original cocoons is comparable to the mass loss of cocoons observed in the field. Variations in the relative abundance of heavy-chain fibrin relative to light-chain fibrin in the separated fibrins were also observed. In the original arsenic, the ratio of heavy-chain to light-chain fibrin was determined to be 1.13 kg/kg. After serine extraction, the ratio was in the range of 0.4-0.8 kg/kg. The reduction in the relative abundance of heavy-chain fibrin indicates the effectiveness of the extraction process in heat-promoting the cleavage of the fibrin chains prior to solubilization and modification. Fibrin Solvent Solubilization and Modification

Controlled solubilization and modification of fibrous proteins is achieved by dispersing the solid protein in a solvent and heat treating the mixture at variable times and temperatures. Typically, a 9.3 M aqueous lithium bromide solution is used as the solvent. The solvent is prepared in a vessel with or without baffles. A center-mounted stirrer with stacked 45° pitch blades is used to mix the solution uniformly into the vessel. Heat transfer oil is circulated through the vessel jacket to stabilize the bulk fluid temperature at the desired reaction temperature while the solvent is mixed. Typically, the reaction temperature is stabilized in the range of 100°C to 103°C (103°C target) or 122°C to 125°C (125°C target).

Once the solvent has reached the desired reaction temperature, the fibrin is loaded into the vessel via an access port in the vessel head. The mass ratio of fibrin to solvent is typically 0.16 kg/kg. Since the density of the solvent significantly exceeds the density of the dried fibrin, it is not obvious that complete dispersion of the fibrin into the solvent is achieved. A substantial downward force is applied to the floating protein pad to completely immerse the material and clear the head space for additional material to be added. Once the head space is cleared, stirring is briefly used to disperse the moistened bulk pad before further additions are made. Loading the vessel with the complete mass of fibrin occurs over a period of 40-60 minutes, during which the reaction temperature is maintained in the vessel.

The reaction time begins after the addition of the fibrin is complete. Stirring is performed throughout the reaction period. The reaction time varies depending on the desired properties of the resulting solution. To produce Activated Silk ^™ 33B (medium MW Activated Silk ^™ ), the reaction is carried out at a working fluid temperature of 100°C to 103°C for 0 to 60 minutes. Figure 76 shows the typical evolution of the average molecular weight of dissolved fibrin as a function of reaction time for Activated Silk ^™ 33B.

To produce Activated Silk ^™ 27P (low MW Activated Silk ^™ ), the reaction was carried out for 40-420 min at a temperature maintained at 122° C. to 125° C. FIG77 shows the typical evolution of the average molecular weight of soluble fibrin as a function of reaction time for Activated Silk ^™ 27P.

The contents of the container are then cooled. Cooling is accomplished by one of two methods. In one method, cooling is performed by immediately removing the solution from the container, dividing the solution into small volumes, and storing the container in a refrigerator at 4°C. In the other method, cooling is performed by recirculating cooled heat transfer oil through the container jacket in place. If jacketed container cooling is used, the temperature can be reduced to below 60°C within the 70 minutes of the overlap of the reaction cycles. Cooling from 60°C to room temperature can be performed more slowly by ambient radiation or by forced refrigeration. When forced refrigeration is used, the solution can be brought to room temperature in 3 hours. Activated Silk ^™ Purification

The cooled reaction mixture is a viscous liquid consisting of water, a stabilizing salt (usually LiBr), fibrin, and other undissolved organic solids. The fibrin must be separated from this mixture. Purification is performed via three filtration stages.

The reaction mixture is first dead-end filtered through needle-wool polypropylene filter media with a nominal particle size range of 1 μm to 200 μm to remove relatively large, undissolved contaminants. The filtered reaction mixture is then transferred through the filter medium to a holding vessel, with or without a baffle, pre-filled with a certain volume of reverse osmosis/deionized (RODI) water. The volume of water fed to the holding vessel is determined by multiplying the volume of the reaction mixture by the water-to-reaction mixture ratio. This ratio ranges from 1 to 7 L/L, depending on the desired product and downstream processing conditions.

Using a center-mounted agitator with stacked 45°-spaced blades or an impeller, mix the reaction mixture with the dilution water until homogeneous. If the diluted material is stored for more than 24 hours, circulate cooled propylene glycol through the container jacket to cool the diluted mixture. Agitation and mixing should be limited to the bare minimum to achieve homogeneity, as excessive or prolonged shear on the fluid increases the risk of product loss due to sedimentation or foaming.

The diluted reaction mixture was subjected to additional dead-end filtration through a melt-blown and spunbonded centrifugally bonded polypropylene medium with a nominal 0.2 μm rejection or a resin-bonded cellulose/diatomaceous earth slow medium with an absolute 2.5 μm rejection to reduce the solution turbidity to below the desired critical limit.

The diluted reaction mixture is transferred through the filtered medium to a tangential flow filtration (TFF) unit equipped with a jacketed retentate container, a rotary toothed pump, a 10 kDa molecular weight cutoff hollow fiber ultrafiltration membrane, and an automatically controlled backpressure valve to stabilize the transmembrane pressure (TMP) during processing. The TMP is defined as the average internal pressure of the TFF unit minus the permeate line pressure. The diluted reaction mixture is recirculated between the retentate container and the membrane contacts via the toothed pump and backpressure valve. The pump is operated to maintain a constant recirculation flow rate, typically in the range of 200 to 500 L/min, depending on the application. Actuate the backpressure valve to maintain the TMP in the range of 7-18 psig, depending on the application. Circulate cooled or heated propylene glycol or water through the retentate vessel to maintain the working fluid temperature between 20°C and 35°C, depending on the application.

Operating under these conditions drives the permeation of water, LiBr and smaller fibrin fragments through the membrane selective layer to the waste. Most of the dissolved fibrin is retained by the membrane. The TFF operation begins with filtration, where the membrane permeate is lost as volume increases and the volume in the retentate vessel is maintained by backfilling with RODI water. Filtration conditions are maintained until the conductivity of the permeate drops below the desired critical value, typically 10 to 50 μS/cm. Critically, the LiBr concentration must be below 150 ppm. Once this condition is met, filtration is stopped, at which point the flow of RODI water to the system stops and the working fluid is allowed to concentrate as osmosis continues at the maintained TMP and flow conditions. Monitor protein concentration during the concentration phase of the operation. Maintain concentration conditions in the range of 5-17% wt, depending on the application. The total residence time in the TFF unit ranges from 12-35 hours, depending on the application.

The purified Activated Silk ^™ solution was removed from the TFF unit and stored in HDPE carbon masonite or stainless steel bags. The Activated Silk ^™ solution was stored at 4°C to maximize shelf life.

Process development has resulted in improved product yields. When the purification train was initially scaled up, yield increased approximately 100-fold for Activated Silk ^™ 27P and approximately 40-fold for Activated Silk ^™ 33B compared to existing processes. As the processing technology matured, yields increased further by approximately 1.75-fold for Activated Silk ^™ 27P and approximately 2.9-fold for Activated Silk ^™ 33B.

Furthermore, process development has demonstrated significant quality improvements by reducing variation in key quality parameters, particularly the weight-average molecular weight and dispersion characteristics of the protein population in the final product. Compared to the previous process, the first scale-up resulted in a 58% reduction in the standard deviation of molecular weight measurements and a 31% reduction in the standard deviation of dispersion measurements for Activated Silk ^™ 27P. Compared to the previous process, the first scale-up also resulted in a 29% reduction in the standard deviation of molecular weight measurements and a 59% reduction in the standard deviation of dispersion measurements for Activated Silk ^™ 33B. Compared to the previous process, the second scale-up resulted in a 64% reduction in the standard deviation of molecular weight measurements and a 12% reduction in the standard deviation of dispersion measurements for Activated Silk ^™ 27P. Compared to the previous process, the second scale-up also resulted in a 75% reduction in the standard deviation of the molecular weight measurement and a 70% reduction in the standard deviation of the dispersion measurement for Activated Silk ^™ 33B.

Fibrin separation requires the separation of serine from the original B. mollissima fibers. This separation is performed in a single stage and facilitated in a solid extraction operation. The unit comprises an atmosphere container enclosing a perforated trough that rotates on a horizontal axis. The raw sugars are loosely packed in permeable mesh bags before the raw fiber is loaded into the rotating drum via a sealable access port. This primary containment minimizes product loss, protects the equipment by preventing the flow-through flake strands from becoming entangled with the rotating components in the container, and protects the drain line from becoming clogged with solids that would otherwise escape from the drum trough during processing.

The vessel is filled with an extraction solvent comprising 0.705% sodium carbonate in water to partially immerse the perforated bubbler. This solvent composition has been shown to effectively dissolve and stabilize serine in solution. The argon/solvent ratio is 0.068 kg/kg. An electric heater located in the base of the vessel is used to maintain the temperature of the extraction solvent in the range of 94.5°C-97°C. Maintaining the extraction solvent at an elevated temperature drives the serine solvent complex. Secondly, the high temperature thermally cleaves the fibrous protein chains to reduce the average molecular weight of the protein population. The rotary drum is periodically rotated during the 30 minute isothermal phase of the extraction. This action serves to expose all fiber surfaces to the extraction solvent.

Then rinse with plenty of hot water. Fill the container with non-perforated water to partially submerge the perforated bubbler. Maintain the rinse water temperature between 55°C and 65°C for 20 minutes, with intermittent bubble rotation, and then drain the rinse water to waste. Repeat this two more times.

After rinsing, the drum rotates at high speed to remove the water retained in the colon by centrifugation. The wet swabs of fibrous protein with a moisture content of 15% to 65% wt. (average 46.74%) are then manually removed from the pads and evenly distributed on perforated trays. The residual moisture is driven off the fibrous protein by storage in a dryer maintained at an internal temperature of 55°C to 60°C until the moisture content of the material is less than 1% of the total weight. Fibrin Solvent Complexation and Modification

Controlled solubilization and modification of fibrin is achieved by dispersing the solid protein in a solvent and heat-treating the mixture at variable times and temperatures. A 9.3 M aqueous lithium bromide solution is used as the solvent.

To produce high MW Activated Silk ^™ , add 400 mL of solvent to two 4 L glass flasks. The flasks are then placed in an oven set at 160°C and heated until the solvent temperature is 100°C ± 4°C. Once the solvent temperature is stable, add 100 g of dried cellulose to each flask and thoroughly wet with solvent by manually stirring with a stainless steel spatula. The flasks are then returned to the oven set at 160°C and kept in the oven for 15-60 minutes, depending on the desired product properties. The solvent temperature is continuously monitored to ensure it remains at 100°C ± 5°C during the reaction period.

After the desired reaction time, remove the flask from the oven. Allow the flask to cool at ambient temperature for ^30-60 minutes. Remove any undissolved solids from the flask. Measure the remaining liquid volume into a sealed container and store in a refrigerator at 4°C.

The cooled reaction mixture is a viscous liquid consisting of water, LiBr, fibrin, and tiny amounts of undissolved organic solids. The fibrin must be separated from this mixture.

First, the reaction mixture is diluted in reverse osmosis/deionized (RODI) water. The diluted mixture is stirred manually in the dilution container, typically a sealable 5-gal BPA-free carbon container, to make it smooth. The volume ratio of reaction mixture to RODI water is 0.0562 mL/mL. The diluted reaction mixture is then dead-end filtered through a stifled glass filter medium with an absolute 0.65 μm particle size rejection to remove fine undissolved solids and reduce solution turbidity.

The diluted reaction mixture is transferred via the filter media to a tangential flow filtration (TFF) unit equipped with a retentate container (typically a sealable 50 L polypropylene carbon), a variable-speed diaphragm pump, a 10 kDa molecular weight cutoff hollow fiber ultrafiltration membrane, and a manually actuated backpressure valve to stabilize the transmembrane pressure (TMP) during processing. The TMP is defined as the average internal pressure of the TFF unit minus the permeate line pressure. The diluted reaction mixture is recirculated between the retentate container and the membrane contacts via the diaphragm pump and backpressure valve. The pump is manually operated to maintain a constant 10 psi pressure drop across the membrane module. The backpressure valve is manually actuated to maintain the TMP at approximately 35 psi.

Operating under these conditions drives the permeation of water, LiBr and smaller fibrin fragments through the membrane selective layer to the waste. Most of the dissolved fibrin is retained by the membrane. The TFF operation begins with filtration, where the volume in the retentate vessel is maintained by backfilling with RODI water as the volume is lost to the membrane permeate. Filtration conditions are maintained until the conductivity of the permeate is below the required critical value, typically 10 to 50 μS/cm. Critically, the LiBr concentration must be below 150 ppm. Once this condition is met, filtration is stopped, at which point the flow of RODI water to the system is stopped, and the working fluid is allowed to continue to concentrate as it permeates under maintained TMP and flow conditions. Monitor protein concentration during the concentration phase of the run. Maintain concentrated conditions until the protein concentration is in the 5-7%wt range.

The purified Activated Silk ^™ solution was removed from the TFF unit and stored in HDPE carbon masonite or stainless steel bags. The Activated Silk ^™ solution was stored at 4°C to maximize shelf life. Example 36: Low and Medium Amino Acid Modification of Silk

The low- and medium-strength polypeptide compositions described herein are unique to pharmaceutically derived and modified polypeptide compositions and, when isolated, exhibit a wide range of behaviors, from extreme self-assembly to exceptional solubility and stability in various buffers over time, as well as a wide range of average molecular weights and polydispersities. These novel pharmacopoeial-derived polypeptide compositions contain unique modified amino acids produced by unique pharmacopoeial processing methods and scales. Strict control over temperature, component concentrations, salt concentrations, physical agitation, and purification allows for the tailoring of unique peptide compositions within the component species at each step of the process to design for specific performance criteria. Some of the resulting polypeptide compositions exhibit biological activity and are potential treatment candidates.

HETs are versatile materials with applications ranging from the development of implantable medical devices to the development of pharmaceutically valuable soluble peptide formulations. The primary challenge with peptides in solution is their tendency to self-assemble and aggregate, making controlling their solubility extremely difficult. Furthermore, the kinetics of gel/film formation cannot be controlled predictably. The novel peptide compositions described herein contain peptide populations that allow for control of their properties and the development of products with predictable and desired properties. Development of low- and medium-strength / modified peptide compositions.

Activated fibers contain a range of peptides with different properties. These are primarily characterized by their molecular weight and polydispersity. To date, no peptide mixtures have been characterized or produced.

A unique large-scale process is used to produce peptide compositions. Low/medium sleds begin their method using sodium carbonate to remove serine at specific ratios of osmotic fluid, sodium carbonate, and water. Multiple wash cycles at different temperatures, 100°C and 60°C, with agitation, are also key to producing specific natural and modified compositions. The filter is then dried to remove water at a specific temperature, thereby maintaining the filter composition. Next, the soft sugar is dissolved in a high concentration of lithium bromide at 103°C and 125°C for 1 and 6 hours, respectively. Time and temperature allow fine-tuning of the degree of post-translational modification, resulting in a unique peptide composition. The filter is then purified to remove the lithium bromide and concentrated. Produces low and medium sled / modified peptide compositions.

The amber pigment was washed with sodium carbonate at 100°C and 60°C and then dried at 60°C. Subsequently, the cosolvent was dissolved in 9.3 M lithium bromide at 103°C for 1 hour, and 9.3 M lithium bromide was dissolved in a low cosolvent at 125°C for 6 hours. This solubilization step not only controls the molecular weight but also the peptide modification that results in the native/modified composition. The trunk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same process, leaving only the native/modified trunk complex in pure aqueous solution. Each unit operation is strictly controlled with respect to temperature, time, concentration, agitation and shearing. Our silk preparations have unique finishes based on the production method.

Dissolution of the degraded flat carbon (see previous section) is performed at high concentrations of dispersible salt (9M LiBr) and extremely high temperatures exceeding 100°C. The only heat treatment that occurs during the manufacturing process described here promotes the deamidation of asparagine and glutamine residues and the oxidation of methionine. From now on, the deamidation of asparagine and glutamine residues and the oxidation of methionine are referred to as "modifications." To determine the extent of amino acid modification during the various preparation methods, an LC/MS method was used (see LC/MS Analysis of Peptides for more details). When the low-sled wires were compared to the produced medium-sled wires, the low-sled wires were found to be more modified than the medium-sled wires (Figures 79A-79C, Table 81). When the manufactured wires were freeze-dried, the same modification trend was maintained; the freeze-dried low-sled wires were more modified than the freeze-dried medium-sled wires (Figures 80A-80B, Table 82). Low-sled wires produced by one factory were more unique and less modified than low-sled wires produced by another factory (Figures 80A-80B, Table 83). When the cabinets were manufactured using a benchtop setup, the resulting wire preparations were less modified than their sled wire counterparts (Figure 82, comparing M Skid Skid Skid and Menchtop Skit). LC/MS analysis of peptides . Materials and reagents: ● Guanidine hydrochloride (GuHCl) (Sigma catalog number G3272-1KG) ● Dithiothreitol (DTT) (ThermoFisher catalog number 20290) ● Iodoacetamide (EtOAc) (Sigma catalog number I1149-5G) ● HPLC-grade water (Chemical Catalog Number W5-4) ● Acetonitrile (ACN) (Fiber Chemical Catalog Number A955-4) ● Formic acid (FA) (Fiber Chemical Catalog Number A117-10X1AMP) ● Trifluoroacetic acid (TFA) (FisherChemical Catalog Number A116-10X1AMP) ● Sodium acetate (Sigma catalog number S5636-250G) Protease ● Trypsin/Lys-C mixture (Promega catalog number V5073) Cell trypsin (Promega catalog number V1061) ● Glu-C (Promega catalog number V1651) Solution ● 6 M GuHCl ● 50 mM DTT (10X) ● 100 mM (10x) ● 50 mM sodium acetate Method for denaturation, reduction, and alkylation

The samples were stored at 4°C until analysis.

For each sample, an aliquot was obtained and mixed with an equal volume of 6 M guanidine hydrochloride (GuHCl) in a new tube. 50 mM dithiothreitol (DTT) was added to a final concentration of 5 mM and the samples were incubated at 60°C for 30 minutes. After a short equilibration period to room temperature, 100 mM iodoacetamide (EtOAc) was added to a final concentration of 10 mM and the samples were incubated in the dark at room temperature for 30 minutes. The reaction was quenched by adding 50 mM DTT to a final concentration of 5 mM DTT. The sample was diluted in 50 mM sodium acetate to give a final concentration of 0.18 M GuHCl. Protease digestion

Using the provided sample concentrations, three aliquots corresponding to 30 μg of total protein were collected in separate tubes. Samples were then treated with enzymes at a ratio of 1:30 (1 μg protein per protease) overnight at room temperature (trypsin) or 37°C (trypsin/Lys-C and Glu-C). Aliquots treated with trypsin/Lys-C and Glu-C were boosted with the same amount of enzyme and incubated the following day at 37°C for 3 hours. The protease reaction was quenched by adding TFA to a final concentration of 1% (v/v). Samples were centrifuged at 14,000 rpm for 10 minutes, and the supernatant was transferred to an HPLC autosampler vial for LC-MS analysis. LC conditions : Column: C18 column (100 μm × 200 mm, 3 μm) ; Mobile phase A : Water with 0.1% formic acid; Mobile phase B : Acetonitrile with 0.1% formic acid; Flow rate: 300 nL/min (micropump) and 15 μL/min (load pump) ; Chromatography time: 60 min ; Gradient: 14% B (0 to 3.3 min); 14% to 30% B (3.3 to 35 min); 30% to 95% B (35 to 37 min); 95% to 80% B (37 to 39 min); 80% B (39 to 41 min); 80% to 14% B (41 to 44 min); 14% B (44 to 60 min) ; Injection volume: 2 μg ; MS conditions:

The full MS acquisition range was 350 to 1600 m/z. The MS method was based on data-dependent acquisition (DDA) of the top 10 ions with an isolation window of 3.0 m/z and a normalized collision energy of 27. Data Acquisition and Analysis

Data were acquired using Thermo Xcalibur ^™ software and analyzed using Thermo Proteome Discoverer ^™ software. To unambiguously assign specific proteins to the identified peptides, at least two unique peptides per protein were required when searching the Bombyx mori database.

For each modification site, all peptides containing that amino acid were classified as modified or unmodified. The percentage of modification was then calculated using the following formula: Calculation of the percentage of amino acid modification at each position along the amino acid sequence of each protein chain.

To determine the percentage of modified amino acids within the sequence of individual protein chains in the preparation, LC/MS analysis was employed. This analytical method not only distinguishes the sequences of all peptides present in solution but also quantifies their individual concentrations. LC/MS methods have the ability to localize amino acid modifications to specific positions within these sequences, a phenomenon induced by the production technique.

After successfully identifying and quantifying all peptides in the mixture, they were aligned with the protein sequences present in spider balls. This alignment process allowed the origin of each peptide along the heavy and light chains of fibrin, as well as the fibrin hexamer (p25). Thus, the exact position of the amino acids was localized along these polypeptide chains.

To calculate the percentage of modified amino acids at specific positions, all peptides containing those amino acids, both modified and unmodified, are quantified. This involves dividing the amount of peptides containing the modified amino acid by the total amount of peptides containing that specific amino acid (modified and unmodified), and then multiplying the result by 100. This yields the percentage of modified amino acids at a given position. See Figure 83. Example 37: Separation of Low-Sludge/Modified Peptide Compositions by Charge and Size Properties

A novel method for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides, is described herein. This novel composition is termed a low-strand/modified polypeptide composition.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, with carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperature to remove residual water, preserving the polypeptide composition. The silk is then dissolved in a high concentration of lithium salt at 125°C for six hours to obtain a low-sludge silk composition. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The low-sludge silk/modified polypeptide composition comprises a population of silk/modified polypeptides having unique properties.

Low-sled silk/modified peptide compositions do not self-assemble at 5 mg/mL. The low-sled silk/modified peptide compositions contain a variety of silk/modified peptide populations; these low-sled silk/modified peptides were fractionated by anion exchange chromatography and size exclusion chromatography to separate the populations based on charge and size. High-resolution separation of five negatively charged silk compositions—AS77, AS78, AS79, AS80, and AS81—was achieved. The average sizes of these silk compositions differed from one another, with AS77 being the largest and AS81 being the smallest. These silk compositions did not self-assemble under conditions promoting self-assembly at 5 mg/mL.

The low-sludge silk/modified polypeptide compositions described herein are novel compositions of silk and modified polypeptides composed of diverse silk polypeptide populations, produced through specialized processing of natural silk produced by silkworms. These silk compositions contain modified amino acid sequences resulting from the silk processing methods and scale. Strict control of temperature, silk concentration, buffer and salt concentrations, physical agitation, and purification allows for the precise development of silk compositions with a variety of performance criteria. Separation of these populations by charge and size reveals novel properties, such as high solubility and stability in solution over time. Purification methods allow the isolation of silk/modified polypeptide compositions that exhibit biological activity and can be used for therapeutic purposes.

Silk is a complex natural biomaterial with potential for a variety of applications, such as the development of implantable medical devices and the development of soluble polypeptide compositions with therapeutic value. In addition, silk peptides have been shown to have anti-genotoxic effects. However, silk in its natural form is insoluble, and silk polypeptide compositions exhibit poor solubility in solution without proper processing and tend to self-assemble and aggregate over time. The kinetics of this self-assembly are unpredictable and highly dependent on the composition of the silk polypeptide/modified composition. Novel silk/modified polypeptide compositions are generated, and silk/modified polypeptide compositions are isolated to identify specific populations within these compositions. The separation process allows for control of the properties of the silk composition and development of products with predictable and desirable characteristics. Production of low-sludge silk / modified polypeptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the silk glue and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 125°C for 6 hours. This dissolution step not only controls the molecular weight but also the polypeptide modifications that produce the native/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the native/modified silk complex and pure water. The temperature, time, concentration, agitation, and shear of each unit operation were strictly controlled. Separation of low-sled / modified polypeptide compositions Separation of AS77-AS81 components of low-sled / modified polypeptide compositions.

To isolate AS77-AS81, the low-sludge silk/modified peptide complex was fractionated using anion exchange chromatography (Q-Sepharose) after the Q-solution was subjected to HiLoad 26/600 Superdex 200 pg size exclusion chromatography (Figures 84, 85A, and 85B). Prior to chromatography, Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The silk was centrifuged and filtered, followed by loading onto a Q-Sepharose column to remove any pre-formed aggregates. The silk complex was loaded onto the Q-Sepharose column, and the flow-through fraction was collected. Negatively charged silk compositions were eluted using a high salt buffer (50 mM Tris, 500 mM CaCl ₂ ). The eluted fractions were collected together and referred to as the Q-eluted fraction. The Q-eluted material was further fractionated by HiLoad 26/600 Superdex 200 pg, where the largest polypeptide compositions were eluted first and each subsequent fraction had a population of lower molecular weight silk compositions ( Figures 84 , 85A , 85B ). Low-sled silk preparation solutions have a characteristic yellow hue. The Q-eluted fraction has a strong yellow hue, while the flow-through fraction is transparent and tends to self-assemble very quickly. Q-eluted silk compositions separated by size exclusion fractionation also have a yellow hue. When silk formulations AS77-AS81 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each silk formulation exhibited different average Mw and different polydispersity (PDI) values (Figures 86A-86B, Table 86). In general, AS77 had the highest Mw (55714 Da), while AS81 had the lowest Mw (28750 Da). The PDI values also showed differential variation. The PDI value for AS77 was relatively low (1.1836), and the PDI value for AS81 was relatively high (1.3648) (Figures 86A-86B, Table 86). The unfractionated low-sled silk had an average Mw of ~19500, indicating that the majority of the peptide population tended to have lower molecular weights than the fractions AS77-AS81. The polydispersity of the unfractionated low-sled silk was approximately 2.2, significantly higher than that of fractions AS77-AS81. This indicates that the unfractionated low-sled silk is composed of a diverse population of peptides compared to fractions AS77-AS81. The AS77-AS81 silk composition was relatively homogeneous by dynamic light scattering and exhibited a gradual size distribution.

In dynamic light scattering analysis (Figures 89A and 89B, Table 85), AS77-AS81 showed relatively uniform but broad peaks, with AS77 having the largest Z-average value (17.16 nm), followed by AS78 (15.14 nm), and so on (Table 85), demonstrating the efficiency of size fractionation separation by Q-elution fractionation. Self-assembly of low- and medium-sized / modified polypeptide compositions and data derived therefrom.

To investigate the stability of the silk/modified peptide complex in solution, self-assembly assays were performed at a concentration of 5 mg/mL.

The absorbance curve at 550 nm for self-assembly analysis is S-shaped and can be described as a logical curve. A typical logical function is:

A _max is the maximum density of the formed gel, k is the self-assembly rate factor (SARF), t _0.5 is the time point at which 50% of the gel has formed, and e is the exponential equation of the specific curve (see Figure MA, the red dashed line is used to better demonstrate how to calculate these factors for the self-assembly experiment).

Another parameter introduced to characterize the tendency of silk to form gels is the self-assembly factor ( _FSAF ), which is:

These parameters were calculated using experimental data from self-assembly assays performed with various novel isolated silk polypeptides and used to characterize their properties (Figures 88A-88B). We focused on four parameters, collectively referred to as self-assembly kinetic factors: self-assembly rate factor (SARF), A _max , t0.5 , and self-assembly factor (SAF) (Figures 88A-88B). SARF shows how quickly silk self-assembles to form a gel after the reaction starts or gelation nuclei form; A _max shows the density of the gel formed after self-assembly is complete, and t0.5 shows the density of the gel formed after the self-assembly reaction reaches a gel density of At the time required for SAF to self-assemble, the filaments showed a tendency to self-assemble (Figures 88A-88B). The AS77-AS81 filament composition did not self-assemble .

Self-assembly analysis showed that the low-sled silk/modified peptide combination did not self-assemble under the experimental system conditions (Figure 89A). No self-assembly occurred after 24 hours, and no self-assembly was detected even 12 days after analysis (Figure 89B). The medium-sled/modified peptide combination served as a positive control and demonstrated rapid self-assembly kinetics (Figures 88A-88B). Materials and methods used to generate and characterize AS77-AS81 silks. Anion exchange chromatography.

Low-sled silk is supplied at a concentration of 60 mg/mL. 50 mM Tris, pH 8.0 buffer is added to the low-sled silk, and the silk is centrifuged at 16,000 rpm (rotor JA-18, Beckman Coulter, average 28,100 × g) for 30 minutes at 4°C to separate the formed aggregates from the soluble silk. The supernatant is collected and filtered through a 0.22 μm PES filter. For silk fractionation, a Q-Sepharose prepacked column connected to an AKTA pure 25 L or HiPrep Q FF 16/10 20 mL column or a HiTrap™ Capto™ Q 1 mL column is used. All buffers used were filtered through 0.22 μm PES filters and degassed by sonication. The centrifuged and filtered low-sludge was loaded onto a 5 × 5 mL HiTrap Q HP column and washed with 10 column volumes of 50 mM Tris pH 8.0, 10 column volumes of 50 mM Tris pH 8.0, 500 mM _CaCl₂ , and finally 10 column volumes of 50 mM Tris pH 8.0. 170 mL of the centrifuged low-sludge was loaded onto the column at a flow rate of 5 mL/min. The flow-through was collected. The column was washed with 50 mM Tris pH 8.0 until the absorbance at 280 nm ( _A₂₀₀ ) reached 100 AU. The bound protein was eluted in a single step using 50 mM Tris pH 8.0, 500 mM CaCl ₂ , and all fractions with an absorbance [A ₂₈₀ ] > 500 AU were pooled. The Q-elution fraction (eluate) was then used for further fractionation by size exclusion analysis .

The low-sled eluate fraction (Q-solubilized) from the Q-Sepharose anion exchange chromatography was used as the starting material for size exclusion chromatography. The filaments were loaded onto a HiLoad 26/600 Superdex 200 pg gel filter column for fractionation using an AKTA Pure 25L system. All buffers used during the fractionation were filtered through a 0.22 μm PES filter and degassed. The low-sled eluate was loaded onto a Superdex 200 gel filter column and run with 50 mM Tris, 200 mM _CaCl₂ , pH 8.0 for fractionation of the Q-solubilized low-sled eluate. The dissolved silk composition was collected in 10 ml fractions. Fractions 6-10 (AS77, AS78, AS79, AS80, AS81) were collected and had a relatively narrow range of molecular weights. The fractions were placed in 3.5 kDa cutoff dialysis bags and concentrated by covering the dialysis bags with polyethylene glycol 35000 Da. The fractions in the dialysis bags were then immersed in 160× the volume of 50 mM Tris pH=8.0 overnight and then immersed in 160× the volume of a new batch of 50 mM Tris pH=8.0. The samples were kept at 4°C until they were used. Analysis / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration is determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation is diluted until the _A280 is between 0.1 and 1. Within this range, absorbance is linearly related to the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution is calculated after adjusting the dilution for absorbance measurement. Analytical size exclusion chromatography.

Analytical size exclusion chromatography was performed as described in detail in EMED-QCP-SILK1-002. Analyses were performed on a 300 mm × 7.8 mm PolySep GFC P-4000 LC column connected to an Agilent 1260 Infinity II HPLC system equipped with an _Agilent G7162A RID refractive index detector. The mobile phase used for the analysis was 0.1 M NaCl, 12.5 mM _Na₂HPO₄ , pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass vial). A 25 μL sample was loaded onto the column and analyzed at 25°C for 20 minutes at a flow rate of 1 mL/min. Molecular weights of various samples were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC&LC/MS Systems and Cirrus SEC data collection and molecular weight analysis software. SDS polyacrylamide gel.

The low-sludge fraction was loaded onto a Mini-Protean TGX precast gel (4-20%), with a protein ladder prestained with Trident marker for molecular weight reference. The SDS-polyacrylamide gel was stained with ReadyBlue ^™ protein staining gel. The gel was immersed in ReadyBlue ^™ solution for 1 hour and then destained with DI/RO water. Self-assembly analysis was performed.

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM CH ₃ COONa pH 5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. First, a buffer solution of 50 mM CH ₃ COONa pH 5.0 and 35% v/v 2-propanol was prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid any air bubbles. Record the absorbance at 550 nm for 24 hours. Export the recorded values to an Excel file for storage and further analysis. Dynamic light scattering analysis of silk compositions.

The low-sludge composition was diluted to a concentration of 1 mg/mL and filtered through a 0.22 μm PES syringe filter. All measurements were performed using a Malvern Zetasizer Pro Red Label with a detection angle of 173°. The Red Label system was operated with a 10 mW He-Ne laser (633 nm). The software used was ZS XPLORER version 3.2.1.11. All measurements were performed using 4.2 ml polystyrene/polystyrene clear cuvettes. Samples were measured at 25°C with an equilibration time of 120 seconds. Intensity magnitude distribution, autocorrelation, and Z-average were measured. Example 38: Separation of Low-Sludge/Modified Peptide Compositions by Size Properties

A novel method for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides, is described herein. This novel composition is termed a low-strand/modified polypeptide composition.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, with carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperature to remove residual water, preserving the polypeptide composition. The silk is then dissolved in a high concentration of lithium salt at 125°C for six hours to obtain a low-sludge silk composition. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The low-sludge silk/modified polypeptide composition comprises a population of silk/modified polypeptides having unique properties.

Low-sled silk/modified peptide compositions do not self-assemble at 5 mg/mL. Low-sled silk/modified peptide compositions contain multiple silk/modified peptide populations; here, the low-sled silk/modified peptides were fractionated by size exclusion analysis, separating the different populations based on size. High-resolution separation of five silk compositions—AS82, AS83, AS84, AS85, and AS86—was achieved. The average sizes of these silk compositions varied, with AS82 being the largest and AS86 being the smallest. These silk compositions did not self-assemble under conditions promoting self-assembly at 5 mg/mL.

In addition to the well-resolved silk compositions, lower molecular weight silk compositions (AS87, AS88, and AS89) were also generated. These compositions exhibited poorer resolution but were composed of significantly smaller peptide populations. These silk compositions self-assembled into gels within days at 5 mg/mL, but were not as rapid as the medium-sled silk compositions (which began self-assembly within 3 hours). Low-sled / modified peptide compositions were separated by size .

The low-sludge silk/modified polypeptide compositions described herein are novel compositions of silk and modified polypeptides composed of diverse silk polypeptide populations, produced through specialized processing of natural silk produced by silkworms. These silk compositions contain modified amino acid sequences resulting from the silk processing methods and scale. Strict control of temperature, silk concentration, buffer and salt concentrations, physical agitation, and purification allows for the precise development of silk compositions with a variety of performance criteria. Separation of these populations by size reveals novel characteristics, such as high solubility and stability in solution over time in some populations, and a tendency to aggregate in others. Purification methods allow us to isolate silk/modified polypeptide compositions that exhibit biological activity and can be used for therapeutic purposes.

Silk is a complex natural biomaterial with potential for a variety of applications, such as the development of implantable medical devices and the development of soluble polypeptide compositions with therapeutic value. In addition, silk peptides have been shown to have anti-genotoxic effects. However, silk in its natural form is insoluble, and silk polypeptide compositions show poor solubility in solution without appropriate processing and tend to self-assemble and aggregate over time. The kinetics of this self-assembly are unpredictable and highly dependent on the composition of the silk polypeptide/modified composition. Novel silk/modified polypeptide compositions are generated, and specific populations are isolated within these compositions. The isolation process allows us to control the properties of the silk compositions and develop products with predictable and desired characteristics. Production of low-strength / modified polypeptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the silk glue and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 125°C for 6 hours. This dissolution step not only controls the molecular weight but also the polypeptide modifications that produce the native/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the native/modified silk complex and pure water. The temperature, time, concentration, agitation, and shear of each unit operation were strictly controlled. Separation of low-sled / modified polypeptide compositions Separation of AS82 -AS89 components of low-sled / modified polypeptide compositions

To isolate AS82-AS89, the low-molecular-weight/modified peptide composition was fractionated using a HiLoad 26/600 Superdex 200 pg size exclusion chromatography column (Figures 90 and 91). Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The silk was centrifuged and filtered, then loaded onto a HiLoad 26/600 Superdex 200 pg column to remove any pre-formed aggregates. The silk composition was separated by HiLoad 26/600 Superdex 200 pg fractionation, with the largest peptide composition eluting first, and each subsequent fraction containing a population of lower molecular weight silk compositions (Figure 91). The solution prepared by low-sled silk preparation has a characteristic yellow hue, and the fractionated silk compositions also have a yellow hue. When silk formulations AS82-AS89 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each silk formulation exhibited different average Mw and different polydispersity (PDI) values (Figure 91, Table 88). Generally, AS82 had the highest Mw (51936 Da), while AS89 had the lowest Mw (6826 Da). PDI values also showed differential variation. AS82 had a relatively low PDI value (1.1738), while AS89 had a higher PDI value (1.3544) (Figures 92A-92B, Table 88). The AS82-AS86 silk composition showed relative homogeneity by dynamic light scattering, while the AS87-AS89 silk composition contained a variety of peptide population sizes .

In dynamic light scattering analysis (Zetasizer Pro, Figures 95A-5C, Table 87), AS82-AS86 showed relatively uniform but broad peaks, with AS82 having the largest Z-average value (18.174 nm), followed by AS83 (15.659 nm), and so on (Table H). AS87, AS88, and AS89 have lower molecular weights and elute later during chromatography, where the resolution of the Superdex 200 is not optimal and does not resolve peptide populations very well (column resolution decreases for proteins with an average size of less than approximately 44 kDa), as can be seen by SDS gel electrophoresis in Figure 93 (fractions 11 and above). Dynamic light scattering showed two peaks in these fractions, indicating the presence of several populations (Figure 95A). Self-assembly of low- and medium-sled / modified polypeptide compositions. Self-assembly analysis and data derived therefrom.

To investigate the stability of the silk/modified peptide complex in solution, self-assembly assays were performed at a concentration of 5 mg/mL.

The absorbance curve at 550 nm for self-assembly analysis is S-shaped and can be described as a logical curve. A typical logical function is: A _max is the maximum density of the formed gel ; k is the self-assembly rate factor (SARF); t _0.5 is the time point at which 50% of the gel has formed; and e is the exponential equation for the specific curve (see Figure SA, the red dashed line is used to better illustrate how to calculate these factors for the self-assembly experiment).

Another parameter introduced to characterize the tendency of silk to form gels is the self-assembly factor ( _FSAF ), which is:

These parameters were calculated using experimental data from self-assembly assays performed with various novel isolated silk polypeptides and used to characterize their properties (Figures 94A-94B). We focused on four parameters, collectively referred to as self-assembly kinetic factors; the self-assembly rate factor (SARF), A _max , t0.5 , and the self-assembly factor (SAF) (Figures 94A-94B). SARF shows how quickly the silk self-assembles to form a gel after the reaction starts or gelation nuclei form; A _max shows the density of the gel formed after self-assembly is complete, and t0.5 shows how quickly the self-assembly reaction reaches a gel density of At the time required for the SAF to self-assemble, the filaments showed a tendency to self-assemble (Figures 94A-94B). The AS82-AS86 compositions did not self-assemble .

Self-assembly analysis showed that the low-sled/modified peptide combination did not self-assemble under the experimental system conditions (Figure 94A). No self-assembly occurred after 24 hours, and no self-assembly was detected even 18 days after analysis (Figure 94B). The medium-sled/modified peptide combination served as a positive control and demonstrated rapid self-assembly kinetics (Figures S94A-94B). The AS87-AS89 combination self-assembled within days .

Self-assembly analysis, as described previously, revealed that AS87-AS89 did not self-assemble within 24 hours (Figure 94A). However, upon incubation for an additional 4-5 days, self-assembly occurred in these filament/modified peptide combinations (Figure 94B). A midsled/modified peptide combination served as a positive control and demonstrated rapid self-assembly kinetics (Figures 94A-94B). Materials and methods used to generate and characterize AS82-AS89 . Size exclusion analysis of filaments.

The starting material, low-sled silk at a concentration of 60 mg/mL, was provided by the manufacturer. The low-sled silk was transferred to 50 mM Tris, pH 8.0 buffer and centrifuged at 16,000 rpm for 30 minutes at 4°C to separate the formed aggregates from the soluble silk. The supernatant was collected and filtered through a 0.22 μm PES filter. The silk was then loaded onto a HiLoad 26/600 Superdex 200 pg gel filter column for fractionation using an AKTA Pure 25 L system. All buffers used during the fractionation were also filtered through a 0.22 μm PES filter and degassed. The low-sled silk was loaded onto a Superdex 200 gel filter column and run with 50 mM Tris, 200 mM _CaCl₂ , pH 8, for fractionation. The eluted silk composition was collected in 10 ml fractions. Fractions 6-10 (AS82, AS83, AS84, AS85, AS86) were collected and had a relatively narrow molecular weight range, while fractions 18-20 (AS87, AS88, AS89) had a broader molecular weight range, as these molecular sizes were outside the HiLoad 26/600 Superdex 200 pg separation range. The fractions were placed in 3.5 kDa cutoff dialysis tubing and concentrated by overlaying the tubing with 35,000 Da polyethylene glycol. The dialysis bag fractions were then immersed in 160× the volume of 50 mM Tris pH 8.0 overnight and then in 160× the volume of a fresh batch of 50 mM Tris pH 8.0. The samples were kept at 4°C until use. Analysis / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration was determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation was diluted until the _A280 was between 0.1 and 1. Within this range, absorbance correlated linearly with the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution was calculated after adjusting the dilution for absorbance measurements. Analytical size exclusion chromatography was performed.

Analytical size exclusion chromatography was performed as described in detail in EMED-QCP-SILK1-002. Analyses were performed on a 300 mm × 7.8 mm PolySep GFC P-4000 LC column connected to an Agilent 1260 Infinity II HPLC system equipped with an _Agilent G7162A RID refractive index detector. The mobile phase used for the analysis was 0.1 M NaCl, 12.5 mM _Na₂HPO₄ , pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass vial). A 25 μL sample was loaded onto the column and analyzed at 25 ^° C for 20 minutes at a flow rate of 1 mL/min. Molecular weights of various samples were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC&LC/MS Systems and Cirrus SEC data collection and molecular weight analysis software. SDS polyacrylamide gel.

The low-sludge fraction was loaded onto a Mini-Protean TGX precast gel (4-20%), with a protein ladder prestained with Trident marker for molecular weight reference. The SDS-polyacrylamide gel was stained with ReadyBlue ^™ protein staining gel. The gel was immersed in ReadyBlue ^™ solution for 1 hour and then destained with DI/RO water. Self-assembly analysis was performed.

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM CH ₃ COONa pH=5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. First, a buffer solution of 50 mM CH ₃ COONa pH=5 and 35% v/v 2-propanol was prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid any bubbles. The absorbance at 550 nm was recorded for 24 h. Dynamic light scattering analysis of silk compositions.

The low-sludge composition was diluted to a concentration of 1 mg/mL and filtered through a 0.22 μm PES syringe filter. All measurements were performed using a Malvern Zetasizer Pro Red Label with a detection angle of 173°. The Red Label system was operated with a 10 mW He-Ne laser (633 nm). The software used was ZS XPLORER version 3.2.1.11. All measurements were performed using 4.2 mL polystyrene/polystyrene clear cuvettes. Samples were measured at 25°C with an equilibration time of 120 seconds. Intensity magnitude distribution, autocorrelation, and Z-average were measured. Example 39: Separation of low-density/modified peptide compositions by charge, hydrophobicity, and size characteristics

A novel method for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides, is described herein. This novel composition is termed a low-strand/modified polypeptide composition.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, with carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperature to remove residual water, preserving the polypeptide composition. The silk is then dissolved in a high concentration of lithium salt at 125°C for six hours to obtain a low-sludge silk composition. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The low-sludge silk/modified polypeptide composition comprises a population of silk/modified polypeptides having unique properties.

The low-density/modified peptide complex did not self-assemble at 5 mg/mL.

Low-sled silk/modified peptide compositions comprise various silk/modified peptide populations. Here, an attempt was made to fractionate these low-sled silk/modified peptides based on charge, hydrophobicity, and size by anion exchange chromatography, followed by hydrophobic interaction chromatography and size exclusion chromatography to separate the different populations. High-resolution separations of ten different low-sled silk/modified peptide compositions were achieved: five negatively charged silk compositions that also exhibited hydrophobic characteristics, namely AS90, AS91, AS92, AS93, and AS94. The average sizes of these silk compositions varied, with AS90 being the largest and AS94 being the smallest. These silk compositions did not self-assemble under conditions promoting self-assembly at 5 mg/mL.

Five other negatively charged silk compositions (AS95, AS96, AS97, AS98, AS99, and AS100) are less hydrophobic than AS90-AS94 and have relatively low molecular weights. These silk compositions have varying average sizes, with AS95 being the largest and AS100 being the smallest. These silk compositions may exhibit a tendency to aggregate in solution, as demonstrated by dynamic light scattering and material loss after filtration through a 0.2 μm PES filter.

The low-sludge silk/modified polypeptide compositions described herein are novel compositions of silk and modified polypeptides composed of diverse silk polypeptide populations, produced by specialized processing of natural silk produced by silkworms. These silk compositions contain modified amino acid sequences resulting from the silk processing methods and scale. Strict control of temperature, silk concentration, buffer and salt concentrations, physical agitation, and purification allows for the precise development of silk compositions with a variety of performance criteria. Separation of these populations by charge, hydrophobicity, and size reveals novel characteristics, such as high solubility and stability in solution over time in some populations, and a tendency to aggregate in others. Purification methods allow us to isolate silk/modified polypeptide compositions that exhibit biological activity and can be used for therapeutic purposes.

Silk is a complex natural biomaterial with potential for a variety of applications, such as the development of implantable medical devices and the development of soluble polypeptide compositions with therapeutic value. In addition, silk peptides have been shown to have anti-genotoxic effects, however, silk is insoluble in its native form, and silk polypeptide compositions exhibit poor solubility in solution without appropriate processing and tend to self-assemble and aggregate over time. The kinetics of this self-assembly are unpredictable and highly dependent on the composition of the silk polypeptide/modified composition. Novel silk/modified polypeptide compositions are generated, and specific populations are isolated within these compositions. The isolation process allows us to control the properties of the silk compositions and develop products with predictable and desired characteristics. Production of low-strength / modified polypeptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the silk glue and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 125°C for 6 hours. This dissolution step not only controls the molecular weight but also the polypeptide modifications that produce the native/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the native/modified silk complex and pure water. The temperature, time, concentration, agitation, and shear of each unit operation were strictly controlled. Separation of low-sled / modified peptide compositions Separation of AS90-AS100 peptide components of low -sled / modified peptide compositions

To separate AS90-AS100, the low-density filament/modified peptide complex was fractionated using anion exchange chromatography (Q-Sepharose chromatography), followed by hydrophobic interaction (HIC) chromatography (Butyl ImpRes column), and then HiLoad 26/600 Superdex 200 pg size exclusion chromatography of the Q-HIC eluate (AS90-94) and Q-HIC flow-through (AS95-100) ( Figures 96 , 97A, 97B ).

Prior to chromatography, Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The silk was centrifuged and filtered, then loaded onto a Q-Sepharose column to remove any pre-formed aggregates. The silk composition was loaded onto a Q-Sepharose column, and the flow-through fraction was collected. A high salt buffer (50 mM Tris, 500 mM CaCl ₂ ) was used to elute the negatively charged silk composition ( FIG. 97A ). The eluted fractions were collected together and referred to as the Q-elution fraction. The Q-flow-through fraction was colorless and tended to aggregate.

The Q-eluted fraction was further fractionated using a Butyl ImpRes column (Figure 97B), which separates peptides based on hydrophobicity. Chromatography was performed in the presence of 300 mM ammonium sulfate [( _NH4 ) _2SO4 ] to expose hydrophobic regions within the silk peptides. The highly charged flow-through fraction (Q-HIC-flow-through) was collected for further fractionation _by size exclusion chromatography. In the absence of ammonium sulfate, 50 mM Tris, pH 8.0, was used to elute the more hydrophobic bound silk peptides, reversing the exposure of hydrophobic regions within the silk peptides, which resulted in their release from the Butyl ImpRes column.

The Q-HIC eluate was further fractionated using a HiLoad 26/600 Superdex 200 pg column, where the largest polypeptide components eluted first, and each subsequent fraction contained a population of lower molecular weight filament components (Figures 96, 97C, and 97D). The separated fractions were AS90-AS94. Subsequently, the Q-HIC flow-through fraction was also fractionated using a HiLoad 26/600 Superdex 200 pg column, resulting in AS95-AS100. Comparing the size exclusion chromatograms of Q-HIC(elute) and Q-HIC(flowthrough) ( FIG. 97E ), it is apparent that the Q-HIC(elute) fraction is composed of a higher molecular weight peptide complex, whereas the silk peptides constituting the Q-HIC(flowthrough) fraction elute later, indicating a smaller molecular weight (see also FIG. 99A and FIG. 99B for SDS-PAGE of fractions AS90-AS94 ( FIG. 99A ) and AS95-AS100 ( FIG. 99B )).

The solution prepared by low-sled filaments has a characteristic yellow hue. The Q-solubilizer and Q-HIC-solubilizer fractions have a strong yellow hue, while the Q-flowthrough fraction is transparent and tends to self-assemble very quickly. The Q-HIC-solubilizer silk composition separated by size exclusion fractionation (AS90-94) also has a yellow hue. The Q-HIC-flowthrough fraction separated by size exclusion chromatography (AS95-AS100) is colorless.

When the filaments AS90-AS94 and AS95-AS100 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each filament exhibited different average Mw and polydispersity (PDI) values (Figures 98A-98B, Table 90). Among the Q-HIC-eluted fractions, AS90 had the highest Mw (54,620 Da), while AS94 had the lowest Mw (21,057 Da). The PDI values also showed differential variation. AS90 had the lowest PDI value (1.1487), while AS94 had an even higher PDI value (1.3615) (Figures 98A-98B, Table 90). The Q-HIC flow-through fraction consisted of smaller silk-peptide populations, with AS95, the first eluted fraction, having the highest molecular weight (45,262 Da) in this fraction, 10 kDa smaller than the first eluted fraction of the Q-HIC flow-through fraction (AS90). AS100 had the lowest molecular weight (22,799 Da) in the Q-HIC flow-through fraction. Generally, AS95-AS100 exhibited a trend toward higher polydispersity compared to AS90-AS94. AS95 had the lowest PDI value (1.1988), while AS100 had a higher PDI value (1.5438) (Figures 98A-98B, Table 90).

The unfractionated low-sled silk had an average Mw of ~19,500, indicating that the majority of the peptide population tended to have a lower molecular weight than the fractions AS90-AS100. The polydispersity of the unfractionated low-sled silk was approximately 2.2, significantly higher than the value for the fractions AS90-AS100. This suggests that the unfractionated low-sled silk is composed of a variety of different peptide populations compared to the fractions AS90-AS100. The AS90-AS94 silk composition was relatively homogeneous by dynamic light scattering and exhibited a gradual particle size distribution.

In dynamic light scattering analysis (Zetasizer Pro, Figures 101A-101F, Table 89), AS90-AS94 exhibited relatively uniform but broad peaks, with AS90 having the largest Z-average value (17.617 d.nm), followed by AS91 (16.803 d.nm), and so on (Table 89), demonstrating the efficiency of size fractionation separation by Q-HIC-solvent fractionation (Figures 101C and 101D). Low and mid sled wires exhibited broad peaks (Figures 101A and 101B), while the AS90 fraction exhibited a more uniform and narrower peak compared to both and the Q-HIC-solvent fraction derived from AS90. The AS95-AS100 silk composition exhibited a heterogeneous silk composition by dynamic light scattering.

Dynamic light scattering analysis of AS95-AS100 revealed two major broad peaks in each fraction, indicating a broad size range for the peptide population (Figures 101E and 101F). The Z-average values for AS95-AS100 were not arranged in descending order like those for AS90-AS94 and were more variable (Table 89). The AS95-AS100 fractions and Q-HIC ( flowthrough ) showed a tendency to aggregate.

During the production of AS95-AS100, a large amount of protein material was lost. By detecting the amount of protein that flowed through the Butyl ImpRes column and did not bind to the column by UV280, a large amount of silk peptides was expected to be found in the solution. However, during size exclusion chromatography, the amount of silk peptides in the resulting fractions was low (Figure 99B). It is assumed that most of the silk peptides have aggregated or have not passed the filtration step of Q-HIC (flow-through) and are lost before being loaded onto the gel filter column. SDS-PAGE of AS95-AS100 (Figure 99B) run immediately after size exclusion chromatography showed different silk peptide populations, arranged in descending order of molecular weight. However, dynamic light scattering analysis performed later showed different size populations. This may indicate the aggregation of silk peptides in fractions AS95-AS100 over time. Self-assembly of low and medium sled / modified polypeptide compositions Self-assembly analysis and data derived therefrom.

To investigate the stability of the silk/modified peptide complex in solution, self-assembly assays were performed at a concentration of 5 mg/mL.

The absorbance curve at 550 nm for self-assembly analysis is S-shaped and can be described as a logical curve. A typical logical function is: A _max is the maximum density of the formed gel ; k is the self-assembly rate factor (SARF); t _0.5 is the time point at which 50% of the gel has formed; and e is the exponential equation for the specific curve (see Figure 100 , the red dashed line is used to better demonstrate how to calculate these factors for the self-assembly experiment).

Another parameter introduced to characterize the tendency of silk to form gels is the self-assembly factor ( _FSAF ), which is:

These parameters were calculated using experimental data from self-assembly assays performed with various novel isolated silk polypeptides and used to characterize their properties (Figure 100). We focused on four parameters, collectively referred to as self-assembly kinetic factors: self-assembly rate factor (SARF), A _max , t0.5 , and self-assembly factor (SAF) (Figure 100). SARF shows how quickly silk self-assembles to form a gel after the reaction starts or gelation nuclei form; A _max shows the density of the gel formed after self-assembly is complete, and t0.5 shows the time it takes for the self-assembly reaction to reach a gel density of At the time required for SAF to self-assemble, the filaments showed a tendency to self-assemble (Figure 100). The AS90-AS94 filament composite did not self-assemble .

Self-assembly analysis showed that the low-sled silk/modified peptide combination did not self-assemble under the experimental system conditions (Figure 100). The medium-sled/modified peptide combination served as a positive control and exhibited rapid self-assembly kinetics (Figure 100). Fractions AS95-AS100 were not tested for self-assembly because the purification process did not yield sufficient silk peptides for analysis. Materials and methods used to generate and characterize AS90-AS100 . Anion exchange chromatography of silk.

Low-sled filaments were provided by the manufacturing team at a concentration of 60 mg/mL. 50 mM Tris, pH 8.0 buffer was added to the low-sled filaments, and the filaments were centrifuged at 16,000 rpm (rotor JA-18, Beckman Coulter, average 28,100 × g) for 30 minutes at 4°C to separate the formed aggregates from the soluble filaments. The supernatant was collected and filtered through a 0.22 μm PES filter. For filament fractionation, a Q-Sepharose prepacked column connected to an AKTA pure 25 L or HiPrep Q FF 16/10 20 mL column or a HiTrap™ Capto™ Q 1 mL column was used. All buffers used were filtered through 0.22 μm PES filters and degassed by sonication. The centrifuged and filtered low-sludge was loaded onto a 5 × 5 mL HiTrap Q HP column and washed with 10 column volumes of 50 mM Tris pH 8.0, 10 column volumes of 50 mM Tris pH 8.0, 500 mM _CaCl₂ , and finally 10 column volumes of 50 mM Tris pH 8.0. 150 mL of the centrifuged low-sludge was loaded onto the column at a flow rate of 5 mL/min. The flow-through was collected. The column was washed with 50 mM Tris pH 8.0 until the absorbance at 280 nm ( _A₂₀ ) reached 100 AU. Bound protein was eluted in _{a single} step using 50 mM Tris pH 8.0, 500 mM CaCl₂, and all fractions with an absorbance [ _A₂₀ ] > 500 AU were pooled. This process was repeated twice (separating a total of 300 ml of low-sludge starting material). The Q-elution fraction (eluate) was then used for further fractionation by hydrophobic interaction analysis (HIA) .

The Q-solution was buffer-exchanged with water and dialyzed against 100 volumes of water. After the buffer exchange, 50 mM Tris pH 8.0 and 300 mM (NH ₄ ) ₂ SO ₄ were added to the Q-solution. A Butyl ImpRes resin column was used to generate AS90-AS100. The Butyl ImpRes column was washed with 10 column volumes of degassed and filtered (0.22 μm) 50 mM Tris pH = 8.0, 300 mM (NH ₄ ) ₂ SO ₄ , 10× column volumes of degassed and filtered (0.22 μm) 50 mM Tris pH = 8.0, and 10× column volumes of degassed and filtered (0.22 μm) 50 mM Tris pH = 8.0, 300 mM (NH ₄ ) ₂ SO _4. Q-solution (200 mL) was used for fractional separation. Q-solution filaments were dissolved in 50 mM Tris pH = 8.0, 300 mM (NH ₄ ) ₂ SO ₄ and loaded onto the Butyl ImpRes column. All unbound protein (flow _{-through fraction) was collected and saved for further analysis. After loading, the Butyl ImpRes column was washed with 10× column volumes of 50 mM Tris pH 8.0, 300 mM (NH₄)₂SO₄ until the} OD₂₂O₀ reached approximately 100 AU. Following the wash step, bound silk molecules were eluted with 1.5× column volumes of 50 mM Tris pH 8.0. The eluate was collected, and the Q-HIC (eluate) and Q-HIC (flow-through) fractions were transferred to a dialysis bag and dialyzed against 3 mM Tris, pH 8.0. Both fractions were concentrated by covering the dialysis tubing with polyethylene glycol 35000 Da and saved for further fractionation by size exclusion analysis .

The hydrophobic interaction chromatography (HIC) eluted fraction (Q-HIC eluate) and flow-through fraction (Q-HIC flow-through) were filtered through a 0.22 μm PES filter to remove pre-formed aggregates and fractionated by size exclusion chromatography. Fractionation was performed using an AKTA Pure 25L system, loading the eluate or flow-through fraction onto a HiLoad 26/600 Superdex 200 μg gel filter column. All buffers used during the fractionation were also filtered through a 0.22 μm PES filter and degassed. The Q-HIC (eluate) or Q-HIC (flowthrough) fractions were loaded onto a Superdex 200 gel filter column and run with 50 mM Tris, 200 mM _CaCl₂ , pH 8.0. The eluted silk composition was collected in 10 ml fractions. Fractions 6-10 (AS90, AS91, AS92, AS93, AS94) of the Q-HIC (eluate) were collected and had a relatively narrow molecular weight range. Fractions 8-13 (AS95, AS96, AS97, AS98, AS99, AS100) were collected during the Q-HIC (flowthrough) fractionation. The fractions were placed in a 3.5 kDa cutoff dialysis tubing and concentrated by over-coating the tubing with 35,000 Da polyethylene glycol. The dialysis bag fractions were then immersed in 160× the volume of 50 mM Tris pH 8.0 overnight and then in 160× the volume of a fresh batch of 50 mM Tris pH 8.0. The samples were kept at 4°C until use. Analysis / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration was determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation was diluted until the _A280 was between 0.1 and 1. Within this range, absorbance correlated linearly with the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution was calculated after adjusting the dilution for absorbance measurements. Analytical size exclusion chromatography was performed.

Analytical size exclusion chromatography was performed as described in detail in EMED-QCP-SILK1-002. Analyses were performed on a 300 mm × 7.8 mm PolySep GFC P-4000 LC column connected to an Agilent 1260 Infinity II HPLC system equipped with an _Agilent G7162A RID refractive index detector. The mobile phase used for the analysis was 0.1 M NaCl, 12.5 mM _Na₂HPO₄ , pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass vial). A 25 μL sample was loaded onto the column and analyzed at 25 ^° C for 20 minutes at a flow rate of 1 mL/min. Molecular weights of various samples were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC&LC/MS Systems and Cirrus SEC data collection and molecular weight analysis software. SDS polyacrylamide gel.

The low-sludge fraction was loaded onto Mini-Protean TGX precast gels (4-20%), with a protein ladder prestained with Trident marker for molecular weight reference. The SDS-polyacrylamide gel was stained with ReadyBlue™ Protein Staining Gel. The gel was immersed in ReadyBlue™ solution for 1 hour and then destained with DI/RO water. Self-assembly analysis was performed.

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM CH ₃ COONa pH 5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. First, a buffer solution of 50 mM CH ₃ COONa pH 5.0 and 35% v/v 2-propanol was prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid any air bubbles. The absorbance at 550 nm was recorded for 24 h. Dynamic light scattering analysis of silk compositions.

The low- and medium-sled wire compositions were diluted to a concentration of 1 mg/mL and filtered through a 0.22 μm PES syringe filter. All measurements were performed using a Malvern Zetasizer Pro Red Label with a detection angle of 173°. The Red Label system was operated with a 10 mW He-Ne laser (633 nm). The software used was ZS XPLORER version 3.2.1.11. All measurements were performed using 4.2 ml polystyrene/polystyrene clear cuvettes. Samples were measured at 25°C with an equilibration time of 120 seconds. Intensity magnitude distribution, autocorrelation, and Z-average were measured. Example 40: Separation of midifilament/modified polypeptide compositions by size characteristics.

A novel method for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides, is described herein. This novel composition is termed a mid-spool/modified polypeptide composition.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, with carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperature to remove residual water, preserving the polypeptide composition. The silk is then dissolved in a highly concentrated lithium salt at 103°C for one hour to obtain a mid-sludge silk composition. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The mid-sled filament/modified peptide composition contains a population of filaments/modified peptides with unique properties. The mid-sled filament/modified peptide composition self-assembles at 5 mg/mL.

The mid-sled filament/modified polypeptide composition comprises a variety of filament/modified polypeptide populations. Here, we attempted to separate the different populations based on size by fractionating the mid-sled filament/modified polypeptides using size exclusion analysis. High-resolution separation of six filament compositions was achieved: AS106, AS107, AS108, AS109, AS110, and AS111. The average sizes of these filament compositions varied, with AS106 being the largest and AS111 being the smallest. These filament compositions self-assembled under self-assembly-promoting conditions at 5 mg/mL.

The silk/modified polypeptide compositions described herein are novel compositions of silk and modified polypeptides composed of diverse silk polypeptide populations, produced through specialized processing of natural silk produced by silkworms. These silk compositions contain modified amino acid sequences resulting from the silk processing methods and scale. Strict control of temperature, silk concentration, buffer and salt concentrations, physical agitation, and purification allows for the precise development of silk compositions with a variety of performance criteria. Separation of these populations by charge and size reveals novel properties, such as high solubility and stability in solution over time. Purification methods allow us to isolate silk/modified polypeptide compositions that exhibit biological activity and can be used for therapeutic purposes.

Silk is a complex natural biomaterial with potential for a variety of applications, such as the development of implantable medical devices and the development of soluble polypeptide compositions with medical value. In addition, silk peptides have been shown to have anti-genotoxic effects. However, silk in its natural form is insoluble, and silk polypeptide compositions show poor solubility in solution without proper processing and tend to self-assemble and aggregate over time. The kinetics of this self-assembly are unpredictable and highly dependent on the composition of the silk polypeptide/modified composition. Ovel silk/modified polypeptide compositions are produced, and specific populations are isolated within these compositions. The isolation process allows the properties of the silk compositions to be controlled and products with predictable and desired characteristics to be developed. Production of sled / modified polypeptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the gelatin and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 103°C for 1 hour. This solubilization step not only controls the molecular weight but also the polypeptide modifications that produce the native/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the native/modified silk complex and pure water. The temperature, time, concentration, agitation, and shear of each unit operation were strictly controlled. Separation of the mid -sled / modified polypeptide composition Separation of the AS106-AS111 components of the mid - sled / modified polypeptide composition.

To separate AS106-AS111, the mid-swirl/modified peptide complex was fractionated using a HiLoad 26/600 Superdex 200 pg size exclusion chromatography column (Figures 102 and 103). Tris was added to the filament preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The filaments were centrifuged and filtered, then loaded onto a HiLoad 26/600 Superdex 200 pg column to remove any pre-formed aggregates. Silk compositions were separated by HiLoad 26/600 Superdex 200 pg fractionation, with the largest polypeptide compositions eluting first, and each subsequent fraction possessing a population of lower molecular weight silk compositions (Figures 104A-104B and 405). The mid-sled silk preparation solution had a characteristic yellow hue, and the fractionated silk compositions had a light yellow hue. When silk formulations AS106-AS111 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each silk formulation exhibited different average molecular weights and different polydispersity index (PDI) values (Figures 104A-104B, Table 92). In general, AS106 had the highest molecular weight (89,297 Da), while AS111 had the lowest molecular weight (35,474 Da). The unfractionated mid-filament had the lowest Mw (29,265 Da), indicating that the majority of peptide populations within the mid-filament had lower molecular weights. PDI values also showed a range of variations. AS106 had a relatively low PDI value (1.2866), while AS111 had a higher PDI value (1.4702) ( Figures 104A-104B , Table 92). The unfractionated mid-filament had the highest PDI value of 1.6985, indicating a broad and diverse range of peptide population sizes. The AS106-AS111 composition contained a variety of peptide population sizes .

In dynamic light scattering analysis (Zetasizer Pro, Figures 107A-107B, Table 91), AS106-AS111 showed multiple peptide size populations with two broad peaks for each fraction, similar to the unfractionated mid-sled silk (Figure 107A). There was a shift in molecular size between the fractions, with AS106 having the largest Z-average value (53.71 d.nm) and the AS111 silk composition having the lowest Z-average value (25.34 d.nm). Despite the fractionation by size exclusion analysis, each fraction contained a range of peptides of different molecular sizes, as can be observed by SDS gel electrophoresis in Figure 105. Dynamic light scattering showed two peaks for these fractions, indicating the presence of several populations (Figure 107A). Self-assembly of low- and medium-sled / modified polypeptide compositions. Self-assembly analysis and data derived therefrom.

To investigate the stability of the silk/modified peptide complex in solution, self-assembly assays were performed at a concentration of 5 mg/mL.

The absorbance curve at 550 nm for self-assembly analysis is S-shaped and can be described as a logical curve. A typical logical function is: A _max is the maximum density of the formed gel ; k is the self-assembly rate factor (SARF); t _0.5 is the time point at which 50% of the gel has formed; and e is the exponential equation for the specific curve (see Figure 106 , the red dashed line is used to better demonstrate how to calculate these factors for the self-assembly experiment).

Another parameter introduced to characterize the tendency of silk to form gels is the self-assembly factor ( _FSAF ), which is:

These parameters were calculated using experimental data from self-assembly assays performed with various novel isolated silk polypeptides and used to characterize their properties (Figure 106). We focused on four parameters, collectively referred to as self-assembly kinetic factors: self-assembly rate factor (SARF), A _max , t0.5 , and self-assembly factor (SAF) (Figure 106). SARF shows how quickly silk self-assembles to form a gel after the reaction starts or gelation nuclei form; A _max shows the density of the gel formed after self-assembly is complete, and t0.5 shows the density of the gel formed after the self-assembly reaction reaches a gel density of At the time required for SAF to self-assemble, the filaments showed a tendency to self-assemble (Figure 106). For all parameter calculations, see Table M. The AS106-AS111 filament composite exhibited high self-assembly characteristics .

Self-assembly analysis revealed that the mid-sled silk/modified peptide combination AS106-AS111 self-assembled efficiently under the conditions of the experimental system (Figure 106). All mid-sled fractions tested formed denser gels in a shorter time compared to the unfractionated mid-sled silk. This finding may indicate that the component that promotes self-assembly has a higher molecular weight, because the unfractionated mid-sled silk contains a large number of lower molecular weight peptides, but the silk combination AS106-AS111 is separated by size fraction and contains a higher molecular weight peptide population (Figures 104A-104B and 105). The low-sled silk was used as a negative control and no self-assembly occurred after 17 hours. Materials and methods used to produce and characterize AS106-AS111 . Size exclusion analysis of silk.

The starting material, 60 mg/mL medium sled silk, was provided by the manufacturer. The medium sled silk was transferred to 50 mM Tris, pH 8.0 buffer and centrifuged at 16,000 rpm (rotor JA-18, Beckman Coulter, average 28,100 × g) for 30 minutes at 4°C to separate the formed aggregates from the soluble silk. The supernatant was collected and filtered through a 0.22 μm PES filter. The silk was then loaded onto a HiLoad 26/600 Superdex 200 pg gel filter column for fractionation using an AKTA Pure 25L system. All buffers used during the fractionation were also filtered through 0.22 μm PES filters and degassed. The mid-sled silk was loaded onto a Superdex 200 gel filter column and run with 50 mM Tris, 200 mM _CaCl₂ , pH 8, for fractional separation. The eluted silk composition was collected in 10 ml fractions. Fractions 5-10 (AS106, AS107, AS108, AS109, AS110, AS111) were collected. The fractions were placed in a 3.5 kDa cutoff dialysis tubing and concentrated by covering the tubing with polyethylene glycol 35,000 Da. The dialysis bag fractions were then immersed in 160× the volume of 50 mM Tris pH 8.0 overnight and then in 160× the volume of a fresh batch of 50 mM Tris pH 8.0. The samples were kept at 4°C until use. Analysis / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration was determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation was diluted until the _A280 was between 0.1 and 1. Within this range, absorbance correlated linearly with the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution was calculated after adjusting the dilution for absorbance measurements. Analytical size exclusion chromatography was performed.

Analyses were performed on an Agilent 1260 Infinity II HPLC system using a 300 mm × 7.8 mm PolySep GFC P-4000 LC column connected to an Agilent G7162A RID refractive index detector. The mobile phase used for the analysis was a solution of 0.1 M NaCl, _{12.5 mM} Na₂HPO₄, pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass vial). A 25 μL sample was loaded onto the column and analyzed at a flow rate of 1 mL/min for 20 minutes at 25°C. Molecular weights of each sample were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC & LC/MS Systems, Cirrus SEC data collection and molecular weight analysis software. SDS polyacrylamide gel.

The mid-filament fraction was loaded onto Mini-Protean TGX precast gels (4-20%), with a protein ladder prestained with Trident marker for molecular weight reference. The SDS-polyacrylamide gel was stained with ReadyBlue™ Protein Staining Gel. The gel was immersed in ReadyBlue™ solution for 1 hour and then destained with DI/RO water. Self-assembly analysis was performed.

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM CH ₃ COONa pH=5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. First, a buffer solution of 50 mM CH ₃ COONa pH=5 and 35% v/v 2-propanol was prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid any bubbles. Record the absorbance at 550 nm for 17 hours. Export the recorded values to an Excel file for storage and further analysis. Dynamic light scattering analysis of silk compositions.

The mid-sled composition was diluted to a concentration of 1 mg/mL and filtered through a 0.22 μm PES syringe filter. All measurements were performed using a Malvern Zetasizer Pro Red Label with a detection angle of 173°. The Red Label system was operated with a 10 mW He-Ne laser (633 nm). The software used was ZS XPLORER version 3.2.1.11. All measurements were performed using 4.2 mL polystyrene/polystyrene clear cuvettes. Samples were measured at 25°C with an equilibration time of 120 seconds. Intensity magnitude distribution, autocorrelation, and Z-average were measured. Example 41: Separation of mesofilament/modified polypeptide compositions by charge and size properties.

A novel method for producing compositions of polypeptides derived from silkworms, comprising both native and modified polypeptides, is described herein. This novel composition is termed a mid-spool/modified polypeptide composition.

The novel production method involves removing the silk resin through several washing steps with organic sodium carbonate salts, with carefully controlled multi-stage temperature cycling and agitation as the first step to form the natural/modified polypeptide composition. Next, the silk is dried under controlled temperature to remove residual water, preserving the polypeptide composition. The silk is then dissolved in a highly concentrated lithium salt at 103°C for one hour to obtain a mid-sludge silk composition. The liquid solution is then filtered and purified to remove the lithium salt, leaving only a solution of the natural/modified silk composition and pure water.

The mid-sled filament/modified polypeptide composition contains filament/modified polypeptide populations with unique properties. The mid-sled filament/modified polypeptide composition self-assembled at 5 mg/mL. The mid-sled filament/modified polypeptide composition contains a variety of filament/modified polypeptide populations; here, an attempt was made to separate the different populations based on charge and size by fractionating the mid-sled filament/modified polypeptides using anion exchange chromatography and size exclusion chromatography. High-resolution separation of five negatively charged filament compositions—AS101, AS102, AS103, AS104, and AS105—was achieved. The average sizes of these filament compositions varied, with AS101 being the largest and AS105 being the smallest. These silk compositions self-assembled at 5 mg/mL under conditions that promote self-assembly.

The silk/modified polypeptide compositions described herein are novel compositions of silk and modified polypeptides composed of diverse silk polypeptide populations, produced through specialized processing of natural silk produced by silkworms. These silk compositions contain modified amino acid sequences resulting from the silk processing methods and scale. Strict control of temperature, silk concentration, buffer and salt concentrations, physical agitation, and purification allows for the precise development of silk compositions with a variety of performance criteria. Separation of these populations by charge and size reveals novel properties, such as high solubility and stability in solution over time. Purification methods allow us to isolate silk/modified polypeptide compositions that exhibit biological activity and can be used for therapeutic purposes.

Silk is a complex natural biomaterial with potential for a variety of applications, such as the development of implantable medical devices and the development of soluble polypeptide compositions with medical value. In addition, silk peptides have been shown to have anti-genotoxic effects. However, silk in its natural form is insoluble, and silk polypeptide compositions show poor solubility in solution without proper processing and tend to self-assemble and aggregate over time. The kinetics of this self-assembly are unpredictable and highly dependent on the composition of the silk polypeptide/modified composition. Novel silk/modified polypeptide compositions are generated, and specific populations are isolated within these compositions. The separation process allows the properties of the silk composition to be controlled and products with predictable and desired characteristics to be developed. Production of sled / modified polypeptide compositions.

The silk was washed with sodium carbonate at 100°C and 60°C to remove the silk glue and then dried at 60°C. The silk was then dissolved in 9.3 M lithium bromide at 103°C for 1 hour. This dissolution step not only controls the molecular weight but also the polypeptide modifications that produce the native/modified silk composite. The silk was then filtered to remove undissolved debris and purified using a 10 kDa cutoff PES hollow fiber membrane and concentrated using the same method, leaving only a solution of the native/modified silk complex and pure water. The temperature, time, concentration, agitation, and shear of each unit operation were strictly controlled. Separation of the AS101-AS105 components of the mid-sled / modified polypeptide composition Separation of the AS101-AS105 components of the mid -sled / modified polypeptide composition

To separate AS101-AS105, the Q-solution was subjected to HiLoad 26/600 Superdex 200 pg size exclusion chromatography, followed by anion exchange chromatography (Q-Sepharose chromatography) to fractionate the mid-sled silk/modified polypeptide complex (Figures 108, 109A, and 109B). Prior to chromatography, Tris was added to the silk preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The silk was centrifuged and filtered, followed by loading onto a Q-Sepharose column to remove any pre-formed aggregates. The silk complex was loaded onto the Q-Sepharose column, and the flow-through fraction was collected. Negatively charged silk compositions were eluted using a high salt buffer (50 mM Tris, 500 mM CaCl2). The eluted fractions were collected together and referred to as the Q-eluted fraction. The Q-eluted material was further fractionated by HiLoad 26/600 Superdex 200 pg, where the largest polypeptide compositions were eluted first and each subsequent fraction had a population of lower molecular weight silk compositions (Figures 110A-110B and 111). The mid-sled silk preparation solution has a characteristic yellow hue. The Q-eluted fraction has a strong yellow hue, while the flow-through fraction is transparent and tends to self-assemble very quickly. The Q-eluted silk compositions separated by size exclusion fractionation also have a yellow hue. When silk formulations AS106-AS111 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each silk formulation exhibited different average Mw and different polydispersity (PDI) values (Figures 110A-110B, Table 95). Generally, AS101 had the highest Mw (60,949 Da), while AS105 had the lowest Mw (32,804 Da). The PDI values also showed variation. AS101 had a relatively low PDI value (1.1347), while AS105 had a higher PDI value (1.3937) (Figures 110A-110B, Table 95). The unfractionated sled silk had an average Mw of 29,265, indicating that the majority of the peptide population tended to have lower molecular weights than the fractions AS101-AS105. The polydispersity of the sled silk from the unfractionated separation was 1.6985, significantly higher than the value for fractions AS101-AS105. This indicates that the sled silk from the unfractionated separation is composed of a variety of different peptide populations, most of which have lower molecular weights, compared to fractions AS101-AS105. Dynamic light scattering analysis of the AS101-AS105 silk composition demonstrated that most of the peptide populations were relatively homogeneous and exhibited a gradual size distribution.

In dynamic light scattering analysis (Zetasizer Pro, Figures 113A-113C, Table 94), AS101-AS105 showed two peaks in each fraction, with the smaller size distribution having a higher intensity than the larger size distribution peak. Compared to the unfractionated separation (Figure 113B), which showed two populations with very similar intensities, the Q-SEC fractionation clearly enriched the population with the smaller hydrodynamic radius, and the separation between the different fractions was effective, as shown by the gradual decrease in size from fraction to fraction (Figures 113A-113B, Table 94). AS101 had the largest Z-average value (21.905 d.nm), followed by AS102 (18.735 d.nm), and so on (Table 94), demonstrating the efficiency of size fractionation separation by Q-elution fractionation. Self-assembly of low- and medium-sized / modified polypeptide compositions Self-assembly analysis and data derived therefrom.

To investigate the stability of the silk/modified peptide complex in solution, self-assembly assays were performed at a concentration of 5 mg/mL.

The absorbance curve at 550 nm for self-assembly analysis is S-shaped and can be described as a logical curve. A typical logical function is: A _max is the maximum density of the formed gel ; k is the self-assembly rate factor (SARF); t _0.5 is the time point at which 50% of the gel has formed; and e is the exponential equation for the specific curve (see Figure 111 , the red dashed line is used to better demonstrate how to calculate these factors for the self-assembly experiment).

Another parameter introduced to characterize the tendency of silk to form gels is the self-assembly factor (FSAF), which is:

Using experimental data from self-assembly analyses of various newly isolated silk polypeptides, these parameters were calculated and used to characterize their properties (Figure 111). Four parameters were focused on, collectively referred to as the self-assembly kinetic factors: the self-assembly rate factor (SARF), Amax, t0.5, and the self-assembly factor (SAF) (Figure 111). SARF shows the rate at which silk self-assembles to form a gel after the reaction begins or gelation nuclei form; Amax shows the density of the gel formed after self-assembly is complete, t0.5 shows the time required for the self-assembly reaction to reach a point where the gel density is A_max/2, and SAF shows the direction of silk self-assembly (Figure 111). AS101-AS105 self-assembled to form gels with different kinetics, with the Q- elution fraction exhibiting poor self-assembly.

The silk compositions AS101-AS105 were tested for their ability to self-assemble and form gels. The starting material used to generate the silk compositions AS101-AS105 was the Q-solubilized fraction (Figure 108), which did not show significant self-assembly (Figure 111). However, the silk compositions AS101-AS105 derived from the Q-solubilized fraction self-assembled to form gels with different kinetics (Figure 111, Table 96). This indicates that separating the higher molecular weight peptide population from the lower molecular weight peptide population in the Q-solubilized fraction allows them to self-assemble to form gels. AS101 achieved the lowest Amax, meaning that the gel formed had the lowest density. The AS105 silk composition had the highest _t0.5 value: the gel took the longest to form, and the gelation core took even longer to form. The collected Q- flow-through fractions self-assembled very quickly and formed the densest gel of all the fractions.

When the silk was separated by anion exchange fractionation, the flow-through fraction (Q-FT) was collected (Figure 109A) and demonstrated spontaneous slow self-assembly at 4°C. In order to compare the self-assembly ability of Q-FT with that of the mid-sled silk and the further fractionated silk compositions, Q-FT was tested in the self-assembly assay. The Q-FT fraction showed remarkable self-assembly ability under the analytical conditions and began to self-assemble in less than 1 hour (Figure 111, Table 96). The density of the formed gel was the highest among all the silk compositions tested. These results indicate that the uncharged/positively charged silk composition/modified peptide separated from the negatively charged population by anion exchange has a much higher self-assembly tendency. Materials and methods used to generate and characterize AS101-AS105 silk by anion exchange chromatography.

The filaments were provided by the manufacturing team at a concentration of 60 mg/mL. A 50 mM Tris, pH 8.0 buffer was added to the filaments, and the filaments were centrifuged at 16,000 rpm (rotor JA-18, Beckman Coulter, average 28,100 × g) at 4°C for 30 minutes to separate the formed aggregates from the soluble filaments. The supernatant was collected and filtered through a 0.22 μm PES filter. For filament fractionation, a Q-Sepharose prepacked column connected to an AKTA Pure 25 L or HiPrep Q FF 16/10 20 mL column was used. All buffers used were filtered through a 0.22 μm PES filter and degassed by sonication. The centrifuged and filtered sample was loaded onto a HiPrep Q FF 16/10 20 mL column. The column was washed with 10 column volumes of 50 mM Tris pH 8.0, 10 column volumes of 50 mM Tris pH 8.0, 500 mM CaCl2, and finally 10 column volumes of 50 mM Tris pH 8.0. 150 mL of the centrifuged sample was loaded onto the column at a flow rate of 5 mL/min. The flow-through was collected. The column was washed with 50 mM Tris pH 8.0 until the absorbance at 280 nm (A280) reached 100 AU. The bound protein was eluted in a single step using 50 mM Tris pH 8.0, 500 mM CaCl2, and all fractions with an absorbance [A280] > 500 AU were pooled. The Q-elution fraction (eluate) was then used for further fractionation by size exclusion analysis .

The Q-Sepharose anion exchange fraction (Q-solubilized) was used as the starting material for size exclusion chromatography. The solubilized fraction was loaded onto a HiLoad 26/600 Superdex 200 pg gel filter column for fractionation using an AKTA Pure 25 L system. All buffers used during the fractionation were filtered through a 0.22 μm PES filter and degassed. The Q-solubilized midi-filament was loaded onto a Superdex 200 gel filter column and run with 50 mM Tris, 200 mM CaCl2, pH 8.0 for fractionation separation. The dissolved silk composition was collected in 10 ml fractions. Fractions 6-10 (AS101, AS102, AS103, AS104, AS105) were collected and had a relatively narrow range of molecular weights. The fractions were placed in 3.5 kDa cutoff dialysis bags and concentrated by covering the dialysis bags with polyethylene glycol 35000 Da. The fractions in the dialysis bags were then immersed in 160× the volume of 50 mM Tris pH=8.0 overnight and then immersed in 160× the volume of a new batch of 50 mM Tris pH=8.0. The samples were kept at 4°C until they were used. Analysis / Protein Characterization Methods. Protein Concentration Determination.

Protein concentration was determined by absorbance at 220 nm or 280 nm. The dissolved silk preparation was diluted until the A280 was between 0.1 and 1. Within this range, absorbance correlated linearly with the silk concentration in solution, with a correlation of 1 AU = 1 mg/mL soluble silk protein. The final concentration in the initial silk solution was calculated after adjusting the dilution for absorbance measurements. Analytical size exclusion chromatography was performed.

Analytical size exclusion chromatography was performed as described in detail in EMED-QCP-SILK1-002. Analyses were performed on a 300 mm × 7.8 mm PolySep GFC P-4000 LC column connected to an Agilent 1260 Infinity II HPLC system equipped with an _Agilent G7162A RID refractive index detector. The mobile phase used for the analysis was 0.1 M NaCl, 12.5 mM _Na₂HPO₄ , pH 7 (pH adjusted with phosphoric acid and filtered through a 0.2 μm PES filter into a clean glass vial). A 25 μL sample was loaded onto the column and analyzed at 25°C for 20 minutes at a flow rate of 1 mL/min. Molecular weights of various samples were calculated using Agilent Technologies Open LAB CDS ChemStation Edition for LC&LC/MS Systems and Cirrus SEC data collection and molecular weight analysis software. SDS polyacrylamide gel.

The mid-filament fraction was loaded onto Mini-Protean TGX precast gels (4-20%), with a protein ladder prestained with Trident marker for molecular weight reference. The SDS-polyacrylamide gel was stained with ReadyBlue™ Protein Staining Gel. The gel was immersed in ReadyBlue™ solution for 1 hour and then destained with DI/RO water. Self-assembly analysis was performed.

Silk self-assembly assays (SAF) were performed in 35% v/v 2-propanol and 50 mM CH ₃ COONa pH=5. Each reaction was performed in a final volume of 200 μL. The total silk protein concentration was 5 mg/mL. First, a buffer solution of 50 mM CH ₃ COONa pH=5.0 and 35% v/v 2-propanol was prepared. DI/RO water was then added so that after adding the required volume of silk protein to reach a final concentration of 5 mg/mL, the total volume was 200 μL. Finally, the protein was added and mixed by very gentle pipetting to reduce shear forces. The protein mixture was carefully placed in the wells of a flat-bottom 96-well plate and a layer of 100 μL of mineral oil to avoid any bubbles. Record the absorbance at 550 nm for 24 hours. Export the recorded values to an Excel file for storage and further analysis. Dynamic light scattering analysis of silk compositions.

The mid-sled composition was diluted to a concentration of 1 mg/mL and filtered through a 0.22 μm PES syringe filter. All measurements were performed using a Malvern Zetasizer Pro Red Label with a detection angle of 173°. The Red Label system was operated with a 10 mW He-Ne laser (633 nm). The software used was ZS XPLORER version 3.2.1.11. All measurements were performed using 4.2 ml polystyrene/polystyrene clear cuvettes. Samples were measured at 25°C with an equilibration time of 120 seconds. Intensity magnitude distribution, autocorrelation, and Z-average were measured. Example 42: Absolute weight average molecular weight and polydispersity of low, medium and high molecular weights by SEC-MALS Overview of molecular weight measurement method

This example discusses the measurement of molar mass moments, specifically Mw, by SEC - MALS. Molar mass is used interchangeably with the term "molecular weight." For clarity, molar mass is used in this example.

The method previously used for molar mass determination was SEC-RI. The following section aims to describe the differences between the two methods and how they may be used in future fills. Since both methods produce reported values for weight average molecular weight (Mw) , it is recommended that Mw values be redefined as "absolute Mw " for SEC-MALS and "relative Mw " for SEC-RI . Summary

SEC is a chromatographic separation mode and does not inherently provide absolute molar masses. Instead, SEC provides relative molar masses of proteins (or polymers) based on a calibration curve against standards; this conventional calibration method provides relative molar masses. SEC is typically paired with a concentration-sensitive, rather than molar mass-insensitive, RI detector.

Additional Information: With SEC, molecules are separated by their hydrodynamic size, which is related to molar mass (larger molecules have shorter retention times than smaller molecules). In this paper, the retention time of the protein of interest is correlated to a calibration curve constructed from proteins or polymers of known molar mass. The unique structure or shape of the outer membrane protein will affect the retention time in SEC; this can lead to incorrectly reported molecular weights, especially if the protein of interest differs significantly from the calibration curve. However, this method is often used as the industry standard for LS detectors, and this type of SEC-MALS has not yet been widely adopted as a single molecular weight determination method. Measuring Molar Mass Moments by SEC-MALS

This example reports molecular weight and polydispersity as measured by size exclusion chromatography-multi-angle light scattering (SEC-MALS) for fractionation profiles. SEC-MALS produces absolute weight-average molar mass (Mw) measurements, as opposed to relative molar mass measurements produced by conventional calibration methods, such as SEC-RI.

This document uses two detectors to calculate the molar mass moments of SEC-MALS: the refractive index (RI) detector and the light scattering (LS) detector. The RI detector provides protein concentration. The LS detector directly and absolutely provides the weight-average molar mass (Mw) of the protein; the intensity of the scattered light is directly proportional to the Mw of the molecule. LS is the most widely used technique for determining Mw. Calculation of molar mass moments

Molar mass calculations use the following variables: n _i , M _i , and c _i . _{M i} and c _i are measured directly by SEC-MALS. Note that the subscript "i" indicates that the value is calculated for each fragment of the peak of interest. ● n _i is the number of molecules ● M _i is the molecular mass of the molecule (measured by the LS detector) ● c _i is the material concentration measured by the concentration detector (measured by the RI detector). The number average Mn of the molar mass is related to the arithmetic mean of the total mass divided by the number of molecules. For weight average molecular weight, Mw: Calculation of Polydispersity Index (PDI)

The ratio of Mw to Mn yields the PDI value, as calculated by ASTRA software. Polydisperse macromolecules have a PDI > 1.05, while monodisperse macromolecules have a PDI < 1.05 (Figure 114). For low molecular weight yarns ( low MW yarns ) , the manufacturing method has minimal effect on Mw but can lead to significant differences in PDI .

The weight-average molecular weight of the low MW yarns ranged from 35.9 to 41.2 kDa (Table 99). The average Mw of the low MW yarns was 38.4 kDa (5% RSD). The PDI measurement showed even more significant variability, with the low MW determination produced using the Benchtop method having the highest PDI (2.051), while the PDIs of the other batches of low MW yarns were approximately 1.5. The polydispersity of the low MW yarns ranged from 1.416 to 2.051. For medium molecular weight yarns ( medium MW yarns ) , the manufacturing method had minimal effect on Mw but could lead to significant differences in PDI .

The weight-average molecular weight of medium MW yarns ranged from 51.8 to 85.5 kDa (Table 99). For medium MW yarns, the average Mw was 73.8 kDa (18% RSD). Medium MW yarns produced on the Benchtop had the lowest Mw. The polydispersity of medium MW yarns ranged from 1.445 to 1.799. Dissolution time is a key factor in determining high molecular weight yarns ( high MW yarns ) .

The high MW filaments, with weight average molecular weights ranging from 67.6 to 95.1 kDa, had longer dissolution times, resulting in materials with lower Mw and higher PDI (Table 99). The PDI was 1.566 and 1.39 at longer and shorter dissolution times, respectively.

Analyses were performed using a PolySep GFC P-4000 LC column, 300 mm x 7.8 mm, connected to an Agilent 1260 Infinity II HPLC system with a Wyatt LS and RI detector (Table 97).

The mobile phase used for the analysis was a 12.5 mM _Na₂HPO₄ , 100 mM NaCl solution titrated _{with H₃PO₄} to a final pH of 7.0 ± 0.2. Prior to installation on the HPLC, the solvent was filtered through a 0.22 μm PES filter into _a clean glass vial. A summary of key method parameters is described in Table 98. Data Analysis

All samples were processed using ASTRA 7.3.2 software according to the supplier's recommended procedures. Reportable values for Mw, Mn, and PDI were derived from ASTRA 7.3.2. Other data analyses and graphical presentations were performed using GraphPad Prism version 9.5.1 and Jane Illustrator version 27.5. Example 43: Absolute Weight Average Molecular Weight and Polydispersity of Low-Sliding/Modified Polypeptide Compositions and Medium-Sliding/Modified Polypeptide Compositions by Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) Development of Medium-Sliding / Modified Polypeptide Compositions

The development of low and medium filament modified polypeptide compositions has been previously discussed for the generation of low and medium filaments and the subsequent isolation of modified polypeptide compositions.

This document discusses the measurement of molar mass moments, specifically Mw , by SEC - MALS. Molar mass is used interchangeably with the term "molecular weight". For clarity, this document will use molar mass.

The method previously used for molar mass determination is SEC-RI. The following section aims to describe the differences between the two methods and how they will be used in future fillings. Because both methods produce reported values for weight-average molecular weight (Mw) , it is proposed to redefine Mw values as "absolute Mw " for SEC-MALS and "relative Mw " for SEC-RI . Alternatively, due to the continuity between fillings, the value derived from SEC-RI can be referred to as Mw without additional qualifiers ("relative").

Summary: SEC (size exclusion chromatography) is a separation mode that does not inherently provide absolute molar masses. Instead, SEC provides relative molar masses of proteins (or polymers) based on a calibration curve against standards; this conventional calibration method provides relative molar masses. SEC is typically paired with a concentration-sensitive, rather than molar mass-insensitive, RI detector.

Additional Information: With SEC, molecules are separated by their hydrodynamic size, which is related to molar mass (larger molecules have shorter retention times than smaller molecules). In this paper, the retention time of the protein of interest is correlated to a calibration curve constructed from proteins or polymers of known molar mass. The unique structure or shape of the outer membrane protein will affect the retention time in SEC; this can lead to incorrectly reported molecular weights, especially if the protein of interest differs significantly from the calibration curve. However, this method is often used as the industry standard for LS detectors, and this type of SEC-MALS has not yet been widely adopted as a single molecular weight determination method. Measuring Molar Mass Moments by SEC-MALS

This paper reports the molecular weight and polydispersity of fractionated proteins measured by SEC-MALS. SEC-MALS produces absolute weight-average molar mass (Mw) measurements, as opposed to relative molar mass measurements produced by conventional calibration methods, such as SEC-RI.

This document uses two detectors to calculate the molar mass moments of SEC-MALS: the refractive index (RI) detector and the light scattering (LS) detector. The RI detector provides protein concentration. The LS detector directly and absolutely provides the weight-average molar mass (Mw) of the protein; the intensity of the scattered light is directly proportional to the Mw of the molecule. LS is the most widely used technique for determining Mw. Calculation of molar mass moments

Molar mass calculations use the following variables: n _i , M _i , and c _i . _{M i} and c _i are measured directly by SEC-MALS. Note that the subscript "i" indicates that the value is calculated for each fragment of the peak of interest. ● n _i is the number of molecules ● M _i is the molecular mass of the molecule (measured by the LS detector) ● c _i is the material concentration measured by the concentration detector (measured by the RI detector). The number average Mn of the molar mass is related to the arithmetic mean of the total mass divided by the number of molecules. For weight average molecular weight, Mw: Calculation of the Polydispersity Index (PDI) PDI is the ratio of Mw and Mn as calculated by the program ASTRA. If PDI > 1.05, the macromolecule is considered polydisperse. Results: Low-density / modified peptide complexes separated by charge and size , AS77-AS81

To isolate AS77-AS81, the low-density filament/modified peptide complex was fractionated using anion exchange chromatography (Q-Sepharose), followed by size exclusion chromatography on a HiLoad 26/600 Superdex 200 pg column. Prior to chromatography, Tris was added to the preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The filaments were centrifuged and filtered, followed by loading onto a Q-Sepharose column to remove any pre-formed aggregates.

The silk composition is loaded onto a Q-Sepharose column and the flow-through fraction is collected. The negatively charged composition is eluted using a high salt buffer (50 mM Tris, 500 mM _CaCl2 ). The eluted fractions are pulled together and called Q-elution fractions. The Q-elution solution is further eluted by HiLoad 26/600 Superdex 200 pg, where the largest polypeptide composition is eluted first, and the following fractions have a population of compositions with lower molecular weight. The low-sled silk preparation solution has a characteristic yellow hue. The Q-elution fraction has a strong yellow hue, while the flow-through fraction is transparent and tends to self-assemble very quickly. The Q-elution silk composition separated by size exclusion fractionation also has a yellow hue. When constructs AS77-AS81 were analyzed by HPLC using analytical SEC-MALS (see Materials and Methods), the weight average molecular weight ranged from 118.2 to 61.1 kDa for AS77, with the highest Mw, and AS81, with the lowest Mw (Figure AL and Table W). There was no strong trend in PDI or fraction, but all were polydisperse (PDI > 1.05). The PDI of the unfractionated LS delaminated fraction was significantly higher than that of AS77-81, indicating that the unfractionated Listeria monocytogenes fraction had a more diverse peptide population. Size-separated low-strand / modified peptide compositions: AS82-AS89

To isolate AS82-AS89, a low-density filament/modified peptide complex was fractionated using a HiLoad 26/600 Superdex 200 pg size exclusion chromatography column. Tris was added to the preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The filament was centrifuged and filtered, then loaded onto a HiLoad 26/600 Superdex 200 pg column to remove any preformed aggregates.

By HiLoad 26/600 Superdex 200 pg fractionation, the largest peptide composition eluted first, followed by subsequent fractions containing lower molecular weight silk composition populations. When constructs AS82-AS89 were analyzed by HPLC using analytical SEC-MALS (see Materials and Methods), each of the constructs exhibited different average Mw and PDI values. By fractionating the low-density silk/modified peptides by preparative size exclusion chromatography (FPLC), the different populations were separated by size; these fractions are AS82-AS89. High-resolution separations of five compositions were achieved: AS82, AS83, AS84, AS85, and AS86. These compositions differ from each other in their average size; AS82 is the largest (100.9 kDa) and AS86 is the smallest (51.5 kDa) of this group (Figures 117A-117B and Table 104). Because the resolution of Superdex 200 is not optimal for separating proteins smaller than 44 kDa, three other fractions, AS87-AS89, were less resolved. Fractions AS87, AS88, and AS89 were significantly smaller than AS82-AS86, with Mw values of 18.2, 18.5, and 11.1 kDa, respectively. This higher polydispersity of AS87-AS89 indicates reduced resolution and is consistent with the sizes measured by DLS. Low-density / modified peptide compositions separated by charge, hydrophobicity, and size : AS90-AS94 and AS95-AS100

To separate AS90-AS100, anion exchange chromatography (Q-Sepharose) was used, followed by hydrophobic interaction (HIC) chromatography. Then, the Q-HIC eluate (AS90-94) and Q-HIC flow-through (AS95-100) were subjected to HiLoad 26/600 Superdex 200 pg size exclusion chromatography to fractionate the low-density/modified peptide complex.

Prior to chromatography, Tris was added to the preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The silk was centrifuged and filtered, followed by loading onto a Q-Sepharose column to remove any pre-formed aggregates. The silk composition was loaded onto a Q-Sepharose column, and the flow-through fraction was collected. The negatively charged composition was eluted using a high salt buffer (50 mM Tris, 500 mM CaCl ₂ ). The eluted fractions were pulled together and designated the Q-flow-through fraction. The Q-flow-through fraction was colorless and tended to aggregate.

Q-elution was further fractionated using a Butyl ImpRes column, which separates peptides based on hydrophobicity. Chromatography was performed in the presence of 300 mM ammonium sulfate to expose hydrophobic regions within the soy pod peptides. The highly charged flow-through fraction (Q-HIC flow-through) was collected for further fractionation by size exclusion chromatography. The more hydrophobically bound peptides were eluted using 50 mM Tris, pH 8.0, in the absence of ammonium sulfate, to reverse the exposure of hydrophobic regions within the peptides, leading to their release from the Butyl ImpRes column.

The Q-HIC eluate was further eluted using a HiLoad 26/600 Superdex 200 pg column. The largest polypeptide components eluted first, and the following fractions contained populations of lower molecular weight components (Table 104 and Figures 119A-119B). The separated fractions were AS90-AS94. Subsequently, the Q-HIC flow-through fraction was also fractionated using a HiLoad 26/600 Superdex 200 pg column, yielding AS95-AS100.

The Q-HIC eluate fraction consisted of a higher molecular weight peptide composition, while the peptides that comprised the Q-HIC flow-through fraction eluted later, indicating a lower molecular weight (Table 105, Figures 119A-119B and 120A-120B). The unfractionated Low Skid protein had an average Mw of 41.2 kDa, indicating that the majority of the peptide population had a lower molecular weight than fractions AS90-AS94 (Table 105). Furthermore, the PDI of the unfractionated separation was significantly higher than that of fractions AS90-AS100 , supporting the view that the unfractionated Low Skid is essentially composed of a very diverse population of peptides (Table 105).

To isolate AS101-AS105, anion exchange chromatography (Q-Sepharose) was used, followed by HiLoad 26/600 Superdex 200 pg size exclusion chromatography of the Q-solution to fractionate the mid-swirl/modified peptide complex. Prior to chromatography, Tris was added to the preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The complex was centrifuged and filtered, followed by loading onto a Q-Sepharose column to remove any pre-formed aggregates. The complex was loaded onto the Q-Sepharose column, and the flow-through fraction was collected. Negatively charged compositions were eluted using a high-salt buffer (50 mM Tris, 500 mM CaCl ₂ ). The eluted fractions were pulled together and designated the Q-elution fraction. The Q-elution was further eluted using a HiLoad 26/600 Superdex 200 pg, where the largest polypeptide compositions were eluted first, and the following fractions contained populations of compositions with lower molecular weights. The solution prepared by the medium sled had a characteristic yellow hue. The Q-elution fraction had a strong yellow hue, while the flow-through fraction was transparent and tended to self-assemble very quickly. The Q-elution composition separated by size exclusion fractionation also had a yellow hue.

When the spiked formulations AS101-AS105 were analyzed by HPLC using analytical SEC-MALS (see Materials and Methods), each spiked formulation exhibited unique average Mw and polydispersity (PDI) values (Figures 121A-121B and Table 106). In general, AS101 had the highest Mw (101.5 kDa), while AS105 had the lowest Mw (48.9 kDa). Unlike Mw, PDI showed no clear trend and was fraction-dependent. The median average Mw of the unfractionated separation was 81.9 kDa, indicating that the majority of the peptide population had a lower molecular weight than the fractionated AS101-AS105. The polydispersity of the sled in the unfractionated separation was 1.697, significantly higher than the value for the fractionated AS101-AS105. This indicates that the unfractionated separation consists of multiple different peptide populations, most of which have lower molecular weights, compared to fractions AS101-AS105.

Note: It has been observed MW Silk ( and subsequent fractions ) will gather over time. SAF The analysis confirmed that the samples were tested several months after the fractionation and this was reflected in the SEC-RI Determination of the comparison SEC-MALS The reported Mw and PDI Value Separate by size / Modified peptide compositions: AS106-AS111

To isolate AS106-AS111, the mid-sled filament/modified peptide complex was fractionated using a HiLoad 26/600 Superdex 200 pg size exclusion chromatography column (Figures 114 and 117A-117B). Tris was added to the preparation to a final concentration of 50 mM Tris-HCl, pH 8.0. The filaments were centrifuged and filtered, then loaded onto a HiLoad 26/600 Superdex 200 pg column to remove any pre-formed aggregates. Fractionation was performed on a HiLoad 26/600 Superdex 200 pg column, with the largest polypeptide composition eluting first, followed by a population of lower molecular weight compositions in subsequent fractions (Figures 119A-119B and 120A-120B). The solution prepared by mid-sled silk had a characteristic yellow hue, and the fractionated silk compositions had a light yellow hue. When the doped formulations AS106-AS111 were analyzed by HPLC using an analytical SEC column (see Materials and Methods), each of the doped formulations exhibited different average molecular weights and polydispersity index (PDI) values (Table 107 and Figures 122A - 122B ). In general, AS106 had the highest molecular weight (204.4 kDa), while AS111 had the lowest molecular weight (67.4 kDa). There is no strong trend between PDI and fraction. The unfractionated medium MW silk diet had the highest PDI of 1.697, indicating a broad and diverse peptide population. Note: It has been observed that medium MW silks ( and subsequent fractions ) will aggregate over time. This was also confirmed by SAF analysis. Samples were tested several months after fractionation, which is reflected in the Mw and PDI values reported by SEC-MALS analysis compared to SEC-RI analysis . Analytical Methods Analytical SEC-MALS

Analyses were performed using a PolySep GFC P-4000 LC column, 300 mm x 7.8 mm (Phenomenex, part number CHO-9229) with a guard column connected to an Agilent 1260 Infinity II HPLC system with a Wyatt LS and RI detector (Table 101).

The mobile phase used for the analysis was a 12.5 mM _Na₂HPO₄ , 100 mM NaCl solution titrated _{with H₃PO₄} to a final pH of 7.0 ± 0.2. Prior to installation on the HPLC, the solvent was filtered through a 0.22 μm PES filter into _a clean glass vial. A summary of key method parameters is described in Table 102. Data Analysis

All samples were processed in ASTRA 7.3.2 according to the supplier's recommended procedures. Reportable values for Mw, Mn, and PDI were obtained from ASTRA 7.3.2. Other data analyses and graphics were performed in GraphPad Prism version 9.5.1 and Jane Illustrator version 27.5.

The ungraded low-sled wire had an Mw of 41.2 and a PDI of 1.575. Example 44: Basecoat and topcoat components for coating systems

The following are examples of amounts of various products that may be included in the topcoat and basecoat according to the coating systems described herein. Example 45: Cellulose-stabilized silicone feel modifier in a semipolar solvent

This article describes the emulsification of a silanol/amino-polysiloxane mixture to prevent water separation and discoloration in leather after sanding as part of the leather finishing process.

While the L0822 topcoat in the L1 coating system offers excellent CFR performance, it lacks a pleasant feel. While existing silicones exist on the market, such as SIL/T99, CERAL FI/62, and many other silicones, they are typically used for polyurethane topcoats. When used in cellulose-based topcoats, they cause discoloration and optical defects. Therefore, there is a need for a silicone-based feel modifier product that delivers a similar "silky" feel to other products without fading after sanding.

There are two main reasons for this incompatibility between currently available silicone feel modifiers and L0822 topcoat products. The first issue is the solvent-based nature of the L0822 topcoat product, whose inherent hydrophobicity translates into poor combability with the water-based silicone emulsions used for common polyurethane topcoats. The second issue stems from the nature of the silicones used in currently available feel modifier products. These silicones are typically based on polydimethylsiloxane or silanols, which are incompatible with cellulose-based films. Both of these issues cause discoloration and other optical issues in the topcoat finish after grinding.

To address this issue, a blend of amine-functionalized polysiloxanes was identified as the optimal polymer blend to deliver the desired feel without discoloring the topcoat. Specifically, among the amine-functionalized polysiloxanes tested, those based on aminoethylaminopropyl performed the best. While some of these are available in aqueous dispersions, only solvent-compatible versions offer the desired performance.

However, this aminoethylaminopropyl-based polysiloxane blend is unstable when exposed to air for extended periods and is intolerant to temperature fluctuations, requiring further stabilization. Commercially available products require a nitrogen blanket to maintain storage stability over extended periods or the use of toxic, highly nonpolar organic solvents to maintain dispersion. This cellulose-stable emulsion was developed to yield an air- and temperature-stable feel modifier that is dispersible in more polar organic solvents and provides a "silky" feel without discoloring the L0822 topcoat. Preparation of A971 Feel Modifier for L1 Cellulose-Based Coating System

First, 200 kDa ethylcellulose (EC) was dissolved in 1-methoxy-2-propanol (MP) at a concentration of 6.07% solids. This solution was mixed using an overhead mixer and a Cowles blade. After the ethylcellulose was completely dissolved in the solvent, Silamine DG-22 (DG-22) supplied by Siltech Corporation was added to the solution at a ratio of 1:7 between DG-22 and EC/MP solution. The final concentration of all components should be 12.5% DG-22, 5% EC, and 82.5% MP. The solution was then mixed using a Cowles blade until the desired viscosity was reached (between 2000 cP and 3000 cP at room temperature).

In the topcoat, the feel modifier can be added at concentrations between 1% and 8%. However, as will be seen in the data below, an optimal concentration of 4% delivers the best feel without compromising topcoat performance.

As can be seen in Table 109, the degree of fading (ΔΔE) did not change with increasing the feel modifier concentration in the topcoat. Furthermore, increasing the feel modifier did improve the feel. At 4%, it offered the best "silky" equivalent of the other available products (SIL/T99, BYK, and Ceral FI/62). Regarding coating performance, after processing, there did not appear to be any impact on barre curl, but CFR did improve with increasing feel modifier concentration.

The optimal concentrations of DG-22 and EC added to the feel modifier to produce a stable top coat without compromising performance were determined to be 5 to 2.5. The final total solids content of the feel modifier should be 17.5%.

Regarding the cellulose concentration in the feel modifier, as shown in Table B, increasing the ethyl cellulose concentration did improve post-sanding discoloration. However, it also increased the viscosity of the feel modifier solution, as shown in Table 5. In particular, at a concentration of 8.5% EC, the solution had such a high viscosity that it did not disperse into the topcoat solution within one hour of top mixing. Regarding feel, the ethyl cellulose concentration had no effect on the feel of the finished product.

Regarding the concentration of DG-22 in the topcoat, as can be seen in Table 111, varying the DG-22 concentration in the topcoat had no effect on the degree of fading. However, reducing the DG-22 concentration did negatively impact the feel of the finished product, which is intuitive given that DG-22 is an active ingredient. This means that the concentration of the feel modifier in the topcoat must be adjusted proportionally to the concentration of DG-22 in the feel modifier to provide the desired feel in the final product. As can be seen in Table 112, the desired feel was achieved when scaled proportionally to the DG22 concentration.

In terms of shelf stability, when varying the concentrations of DG-22 and EC, the optimal concentration in the feel modifier was 12.5% DG-22 and 5% EC. This formulation delivered a stable, clear solution after two weeks at room temperature (Table 114) while also delivering more DG-22 to the topcoat than the 6.25% DG-22 formulation. From an EC perspective, 5% EC provided better short-term stability than 2.5% without becoming cloudy or experiencing a dramatic increase in viscosity (as seen in Tables 114 and 113).

At room temperature, no separation occurred in the feel modifier solution after one month, however, the DG-22 product itself separated without a nitrogen blanket after only two days. Furthermore, after one week at room temperature, the DG-22 product formed an unusable gel, while no change was noted in the feel modifier. Under heat-accelerated aging, the feel modifier remained stable and clear after 32 days at 40°C, while DG-22 itself gelled after only four days. Regarding viscosity changes, after two weeks, the viscosity of the feel modifier solution decreased by 5.8% at room temperature and by 9.9% at 40°C.

When testing temperature stability, the combination of a methoxypropanol solvent and an ethyl cellulose emulsion stabilizer improved stability. At a higher temperature of 50°C, the addition of methoxypropanol by itself had no effect on solution stability or the effectiveness of the additive in the topcoat. At extreme temperatures (10°C and 50°C), the solution gelled or separated, both with and without methoxypropanol, making it less compatible with the dispersion in the topcoat. The addition of ethyl cellulose appeared to preserve additive stability and performance on the leather, particularly its feel and even appearance. Prevents phase changes (gelation or separation) or optical changes (clouding) at low (2.5% EC) and high (5% EC) concentrations added to the additive.

Silamine DG-22 was chosen as the primary form of silicone because it exhibited the least discoloration after grinding. As can be seen in Table 117, it exhibited the least discoloration of all the amine-functional silicones after grinding. Example 46: Plasticizer System for Activated Silk™ L1 Biocoating System

Polyurethane dispersions (PUDs) can be used as plasticizers in solvent systems to prevent cracking in leather after grinding as part of the leather finishing process. Grinding is a key and common practice in tanneries to soften leather after finishing. Grinding is a mechanical softening process in which the leather is tumbled in a drying drum at controlled temperature and humidity. Grinded leather often offers a better feel and ease of processing for certain applications.

While acceptable softness depends on the end application, the minimum viable product (MVP) for the Activated Silk™ L1 Biocoat System is set at 5 hours; achieving 10 hours of grinding is strongly recommended. The Activated Silk™ L1 Biocoat System does not exhibit sufficient softening due to the rigidity of the cellulose-based topcoat, which is designed to provide both dry and wet abrasion resistance. Therefore, a plasticizer or plasticizing material is required to provide adequate softening after grinding.

Many known plasticizers, either as single components or as multi-components, have been tested as film casts and on leather at various concentrations (1.25-20% relative to the solids content of the top coat) (Table 118) with a 2.5% top coat (hereinafter, percentages are expressed relative to the solids content of the top coat). Although they form soft films when cast, they fail during grinding. Failure is defined as cracks, white lines, spots, or sometimes discoloration on the leather surface (Figure 128). At lower concentrations of plasticizer (1.25-5%), the coating on the leather remains hard and cracks and lines begin to form after grinding. However, at higher concentrations (10-20%) of plasticizer, the coating may become too soft and the film may lose its integrity and sometimes fail to form a film. The coating may also delaminate or tear easily after grinding. In addition to cracks, lines, or delamination, using large amounts of plasticizer may result in a very oily coating, which is unacceptable in some applications. In addition, other performance tests such as color fastness to rubbing, deflection, tape, or water drop tests must be performed at the required levels. Table 119 shows some examples of failed systems for known plasticizers and various concentrations.

In addition to conventional plasticizers, PUDs (polyurethane-based adhesives) traditionally used in waterborne systems were also evaluated. There are three main types of PUDs: polyesters, polyethers, and polycarbonates. Polyurethanes are copolymers composed of soft and hard segments. The soft segments, which impart flexibility and elasticity, are typically polyether or polyester polyols, while the hard segments, which provide strength, are made from diisocyanates and chain extenders. The type and amount of soft segments have been shown to play a key role in the crystallinity of polyurethanes.

Because the L1 topcoat is delivered in an organic solvent, the most important requirement for a PUD candidate is that it must be soluble/dispersible and sprayable in the solvent system. Some PUDs are insoluble/dispersible/sprayable, or even unsprayable, even at limited concentrations. Even after meeting the first requirement in the topcoat solvent, some PUDs may fail the milling process (Table 120). Various PUDs were tested at concentrations of 0.5-2.5% solids. Using available information on the PUD type from the supplier, the general types of PUDs that failed were polyester, polycarbonate, or blends of polyester and/or polycarbonate. It should be noted that although polycarbonate-based PUDs have great water and chemical resistance, they have limited flexibility and cannot be ground.

Compared to polyester-based PUDs, polyether-based PUDs offer greater flexibility by inhibiting polymer crystallization due to flexible ether bonds. Furthermore, dispersing PU in water can be achieved through the use of external emulsifiers or by incorporating emulsifying segments into the PU backbone. The latter can be provided by polyether groups, which possess ionic and hydrophilic regions, providing high stability to the dispersion compared to non-polyether derivatives. Furthermore, polyether-based PUDs exhibit superior hydrolysis resistance compared to their polyester counterparts, due to the susceptibility of the ester groups to hydrolysis.

The Activated Silk™ L1 Biocoat system utilizes a composite system of polyether-based PUD and Activated Silk™ as a basecoat and a cellulose-based topcoat. Polyether-based PUD/Activated Silk™ solutions have previously been observed to be highly compatible with leather and cellulose-based topcoats. While PUDs are water-based and typically offer low rubfastness performance and fail water drop tests, the synergistic effect of the composite of polyether-based PUD and the Activated Silk™ L1 Biocoat system provides a high-performance system at low solids concentrations, enabling thin application for improved tactility. How to prepare plasticizer for L1 Biocoating System:

Dilute 5% Activated Silk™ L1 Biocoat System Topcoat (L0822) to 2.5% with an alcohol (ethanol, isopropyl alcohol, methoxypropanol, etc.). Add the polyether-based PUD or polyether-based PUD blend to this solution at a concentration of 0.5-2.5%. Then, mix the solution with a Cowles blade, paddle, or stir bar until uniform, depending on the batch size. Polyether-based PU blends provide different tactile properties:

Another clear requirement for coatings is to achieve tactile properties, such as softness. Softness is generally defined as the signal detected by nerve receptors in the skin when a surface comes into contact with the surface due to the friction between the two surfaces. Because instrumental measurement of the softness of a coating is challenging, the most common technique remains individual assessment of the coating by hand. The softness characteristics of a polyether-based PUD plasticizer in the Activated Silk™ L1 biosystem were also investigated after grinding. Note that not all polyether-based PUDs are created equal (Table E). For example, while HEIM 3317 gave the softest feel, PU 2450 gave the softest. It's speculated that the film's parameter known as "100% modulus" may be the cause ( based on limited data from the supplier ). 100% modulus is defined as the force required for a short elongation. The lower the 100% modulus, the softer the feel.

Blending can also help synergistically improve the softness of the lowest iron. Adding a small amount of the softest iron to the lowest iron (PU 2450) significantly improves the feel of the lowest iron. For example, a blend of Biopur 5000 and HEIM 3317 provides a smoother feel than Biopur 5000 alone. Another finding is that processing conditions have an impact on softness. As the ironing temperature increases, the feel changes from dry and smooth to soft and smooth.

The efficacy of the present invention is that even at low concentrations, the performance of polyether-based PUD and blends is still better than that of conventional plasticizers and can provide a range of tactile properties through different processing conditions. Example 47: Matting Agent 1 -component Matting Agent System

This is Formulation A with several variations: ● One-component formulation ● Addition of hydrophilic fumed silica for stability

Without wishing to be bound by any particular theory, it is believed that replacing triacetin with diacetin makes it more miscible, allowing it to be made as a single component. Example 48: Activated Silk™ Paper Peel Transfer Leather

Figure 129 shows the process of creating a pattern on leather using paper peeling. Biodegradable chemicals are used for the pre-skin layer, skin layer, base coat, and top coat. The release paper is textured and acts as a mold for the final desired pattern. The pattern is carried by the base coat and top coat layers. The "pre-skin" layer is applied to the leather, and the "skin" layer is applied to the leather and dried. Both can be applied by spraying or roller coating. The pre-skin and skin layers are then pressed together using temperature and pressure using a rotary press. The release paper is used to apply the L1 base coat, which is then dried. The L1 top coat is then applied by spray or roll coater and dried. This forms the basecoat/topcoat combination. A rotary press can be used in either of these steps, if desired. The Activated Silk™ top coat has a lower solids concentration than traditional top coats (1.5-3% vs. 15-35%). When applied to the Activated Silk™ L1 basecoat, it forms a composite. Both characteristics enable better flexibility and, ultimately, adhesion to the underlying layer (see Figures 131A and 131B). Example 49: Alternative Activated Silk™ Paper Release Transfer for Leather with Large Defects or Blemishes

Figure 130 shows the process used to create a pattern on leather for leather with large defects or flaws. Biodegradable chemicals can be used for the plaster, front skin layer, skin layer, base coat and top coat. The release paper is textured and acts as a mold for the final desired pattern. The pattern is carried by the base coat and top coat. For large defects or corrected leather that requires texture reconstruction or correction, the plaster can be applied before the skin layer. Both can be applied by spraying or roller. The front skin layer and skin layer are then pressed together using temperature and pressure using a rotary press. The release paper is used to apply the L1 base coat, which is then dried. The L1 top coat is then applied by spray or roll coater and dried. The base coat/top coat composition/composite is then formed. A rotary press can be used in any of the above steps as needed. The layers of skin (pre-skin and epidermis) are pressed to fill in the defects in the leather. The solids concentration of the Activated Silk™ top coat is lower than that of a traditional top coat (1.5-3% vs. 15-35%). When applied to the Activated Silk™ L1 base coat, it produces a composite. Once applied to the Activated Silk™ L1 base coat, it produces a composite as shown in the previous Example 31 (SEM image of a leather sample tested with BSE (SE2)). Both features enable greater flexibility and ultimately adhesion to the underlying layer (see Figures 131A and 131B).

All patents, patent applications, and published references cited herein are incorporated herein by reference in their entirety. Although the methods of the present invention have been described in conjunction with specific embodiments thereof, it will be understood that they are capable of further modification. Additionally, this application is intended to cover any variations, uses, or adaptations of the methods of the present disclosure, including such variations, uses, or adaptations as come within known or customary practice in the art to which the methods of the present disclosure pertain and which are independent of the present disclosure .

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The embodiments disclosed herein will be further explained with reference to the accompanying drawings. The drawings are not necessarily to scale, but emphasis is generally placed on illustrating the principles of the disclosed embodiments. [ Figure 1 ] is a flow chart illustrating various embodiments of the present disclosure for producing pure fibrous protein fragments (SPF). [ Figure 2 ] is a flow chart illustrating various parameters that can be modified during the extraction and dissolution steps during the process for producing the disclosed SPF. [ Figure 3 ] illustrates the general steps used in leather processing. Figure 4 shows photographs of felt pads (and associated leather samples) after 600 consecutive cycles of wet Veslic rubbing, comparing leather samples treated with the fibrous protein fragment composition (bottom sample - entry B2) and leather samples treated with polyurethane (top two samples). Note that after 600 cycles, the polyurethane sample deteriorated and dye leached from the leather into the felt. Figure 5 shows photographs of felt pads after 10 cycles of wet Veslic rubbing on leather samples treated with entries A1, A2, B1, and B2 (Table 1). [ Figure 6 ] is a photograph of a water droplet placed on a sample treated with either the warp fibroin fragment or cross-linked polyurethane coating system after a wet Veslic rubbing. In the case of the fibroin fragment (entry B2), the sample was exposed to 600 rubbing cycles, while the polyurethane sample was subjected to only 10 cycles. The photograph was taken 5 minutes after the water droplet was placed. Note that when using the commercial reference system designed as a top coat, water penetrated into the leather matrix. [ Figures 7A-7B ] are graphical analyses showing the results of Water Vapor Transmission Test #1 on coated leather (7A) and uncoated leather (7B). [ Figures 8A-8B ] are graphical analyses showing the results of Water Vapor Transmission Test #2 on coated leather (8A) and uncoated leather (8B). [ Figures 9A-9B ] are graphical analyses showing the results of Water Vapor Transmission Test #3 on coated leather (9A) and uncoated leather (9B). [ Figure 10 ] is a photograph of uncoated, conventional leather. [ Figure 11 ] shows an FTIR analysis of uncoated, conventional leather. [ Figure 12 ] is a photograph of leather treated with an adhesive coating of the coating system disclosed herein. [ Figure 13 ] shows an FTIR analysis of leather treated with an adhesive coating of the coating system disclosed herein. [ Figure 14A ] is a photograph of leather treated with a top coating layer of the coating system disclosed herein. [ Figure 14B ] shows FTIR analysis of leather treated with a top coating layer of the coating system disclosed herein. [ Figure 15A ] is an IR spectrum of a leather sample treated with the coating system disclosed herein using an LN-MCT detector. [ Figure 15B ] shows macroscopic ATR imaging of a leather sample treated with an adhesive base coating layer of the coating system disclosed herein. [ Figure 15C ] shows macroscopic ATR imaging of a leather sample treated with a top coating layer of the coating system disclosed herein. Figures 16A-16H are photographs showing the results of stain removal tests on leather treated with the coating system disclosed herein using various stain sources. 16A: Mud, 16B: Water, 16C: Mustard, 16D: Corn Oil, 16E: Wine, 16F: Ketchup, 16G: French Dressing, 16H: Coffee. Figures 17A-17C are photographs of leather samples treated with the coating system disclosed herein used in industrial trials. Figures 18A-18I are photographs of a felt pad (and associated leather sample treated with the coating system disclosed herein) after 600 consecutive wet Veslic abrasion cycles (note: Figure 18H was subjected to only 360 cycles). [ Figures 19A-19D ] are photographs showing the results of the Barre deflection test performed on various leather samples treated with the coating system disclosed herein. [ Figures 20A-20I ] are photographs showing the results of the tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [ Figure 21 ] is a photograph showing the difference between leather samples treated with the adhesive coating system disclosed herein before and after abrasion. [ Figures 22A-22I ] are photographs showing the results of the tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [ Figure 23 ] is a photograph showing the difference between leather samples treated with the adhesive coating system disclosed herein before and after grinding. [ Figures 24A-24B ] are photographs showing the difference between leather samples treated with the adhesive coating system disclosed herein before and after grinding in a tape test. [ Figures 25A-25C ] are microscope cross-sectional images of a leather surface treated with the coating system disclosed herein. [ Figures 26A-26C ] are microscope top-view images of a leather surface treated with the coating system disclosed herein. [ Figures 27A-27C ] are images showing a wet blue leather strip treated with the coating system disclosed herein under a digital microscope. 27A: Side view, 27B: Top grain view, 27C: Skin view. [ Figures 28A-28C ] show images of a paper strip treated with the coating system disclosed herein, taken under a digital microscope. 28A: Top view, 28B: Side view, 28C: Back view. [ Figures 29A-29C ] show images of a fabric strip treated with the coating system disclosed herein, taken under a digital microscope. 29A: Top view, 29B: Side view, 29C: Back view. [ Figures 30A-30C ] show images of a fabric strip with a blue tape treated with the coating system disclosed herein, taken under a digital microscope. 30A: Top view, 30B: Side view, 30C: Back view. [ Figure 31 ] Images showing the tensile testing procedure for AS-104 + 2% glycerol + 50 mM magnesium sulfate films. [ Figure 32 ] A suggested mixing mechanism for incorporating AS-104, 2% glycerol, and varying salt concentrations. [ Figure 33A ] Elongation at break for AS-104, 2% glycerol, and guanidine hydrochloride (5, 10, 25, and 50 mM). [ Figure 33B ] Ultimate tensile strength for AS-104, 2% glycerol, and guanidine hydrochloride (5, 10, 25, and 50 mM). [ Figure 34A ] Elongation at break for AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM). Figure 34B shows the ultimate tensile strength of AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM). Figure 35A shows the elongation at break of AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM). Figure 35B shows the ultimate tensile strength of AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM). Figure 36A shows the elongation at break of AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM). Figure 36B shows the ultimate tensile strength of AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM). Figure 37A shows the elongation at break of AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM). Figure 37B shows the ultimate tensile strength of AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM). Figure 38A shows the elongation at break of AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM). Figure 38B shows the ultimate tensile strength of AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM). Figure 39A shows the elongation at break of AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM). [ Figure 39B ] shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM). [ Figure 40A ] shows the elongation at break of AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM). [ Figure 40B ] shows the ultimate tensile strength of AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM). [ Figure 41A ] shows the elongation at break of AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM). [ Figure 41B ] shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM). [ Figure 42A ] Shows the elongation at break of AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM). [ Figure 42B ] Shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM). [ Figure 43 ] Compilation of all data related to elongation at break. [ Figure 44 ] Compilation of all data related to ultimate tensile strength. Figure 45 shows the results of Veslic wet and dry tests on Bodin-based black leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. Figure 46 shows the results of Veslic wet and dry tests on Bodin brown leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. Figure 47 shows the Veslic scores of Bodin-based black leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. Figure 48 shows the Veslic scores of Bodin Brown leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. Figures 49A and 49B show the before-and-after topographic traces of a leather sample coated with GG silk before (Figure 23A) and after (Figure 23B) point-filling with silk + 0.5 wt% GG. The traces were captured using a Taylor Hobson CCI HD optical profiler. [ Figure 50 ] Schematic diagram showing the preparation of a two-part matting agent system. [ Figure 51A ] Gray, brown, and black leathers with CAP-7. [ Figure 51B ] Gray, brown, and black leathers with MHG. [ Figure 51C ] Gray, brown, and black leathers with DPGDB. [ Figure 52 ] Relationship between the weight of the coated sample and the nozzle diameter. [ Figure 53A ] The effect of 2.5% ethyl cellulose in methoxypropanol on unrefined leather. [Figure 53B ] The effect of 2.5% ethyl cellulose in methoxypropanol on refinished leather. [ Figure 53C ] The effect of 5% ethyl cellulose in methoxypropanol on unrefined leather. [ Figure 53D ] Shows the effect of 5% ethyl cellulose in methoxypropanol on refinished leather. [ Figure 53E ] Shows the effect of 7% ethyl cellulose in methoxypropanol on unrefined leather. [ Figure 53F ] Shows the effect of 7% ethyl cellulose in methoxypropanol on refinished leather. [ Figure 54 ] Shows the test results of the matting test of Formulation A. [ Figure 55A ] Shows the samples using Euroleather (top) and Fragopel (bottom) with Formulation A before ironing. [ Figure 55B ] Shows the samples using Euroleather (top) and Fragopel (bottom) with Formulation A before ironing. [ Figure 55C ] Shows the samples before ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55D ] Shows the samples before ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55E ] Shows the samples before ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55F ] Shows the samples before ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55G ] Shows the samples before ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55H ] Shows the sample after ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55I ] Shows the sample after ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55J ] Shows the sample after ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55K ] Shows the sample after ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55L ] Shows the sample after ironing using Euroleather (top) and Fragopel (bottom) with Formula A. [ Figure 55M ] Shows samples after ironing using Euroleather (top) and Fragopel (bottom) using Compound A. [ Figure 55N ] Shows samples after ironing using Euroleather (top) and Fragopel (bottom) using Compound A. [ Figure 56A ] Shows samples after (top) and before (bottom) grinding using Fragopel using Compound A. [ Figure 56B ] Shows samples after (top) and before (bottom) grinding using Fragopel using Compound A. [ Figure 56C ] Shows samples after (top) and before (bottom) grinding using Fragopel using Compound A. [ Figure 56D ] Shows samples after (top) and before (bottom) grinding using Fragopel using Compound A. [ Figure 56E ] Shows the samples after (top) and before (bottom) grinding using Fragopel with Formulation A. [ Figure 56F ] Shows the samples after (top) and before (bottom) grinding using Fragopel with Formulation A. [ Figure 56G ] Shows the samples after (top) and before (bottom) grinding using Fragopel with Formulation A. [ Figure 56H ] Shows the samples after (top) and before (bottom) grinding using Euroleather with Formulation A. [ Figure 56I ] Shows the samples after (top) and before (bottom) grinding using Euroleather with Formulation A. [ Figure 56J ] Shows the samples after (top) and before (bottom) grinding using Euroleather with Formulation A. [ Figure 56K ] Shows the Euroleather sample using formulation A after grinding (top) and before (bottom). [ Figure 56L ] Shows the Euroleather sample using formulation A after grinding (top) and before (bottom). [ Figure 56M ] Shows the Euroleather sample using formulation A after grinding (top) and before (bottom). [ Figure 56N ] Shows the Euroleather sample using formulation A after grinding (top) and before (bottom). [ Figure 57A ] Shows the Euroleather (left) and Fracopel (right) samples using formulations A, B, C, and D before ironing. [ Figure 57B ] Shows the Euroleather (left) and Fracopel (right) samples using formulations A, B, C, and D before ironing. [ Figure 57C ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D before ironing. [ Figure 57D ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D before ironing. [ Figure 57E ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing. [ Figure 57F ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing. [ Figure 57G ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing. [ Figure 57H ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing. [ Figure 57I ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing (top) and grinding (bottom). [ Figure 57J ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing (top) and grinding (bottom). [ Figure 57K ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing (top) and grinding (bottom). [ Figure 57L ] Shows samples of Euroleather (left) and Fracopel (right) using formulations A, B, C, and D after ironing (top) and grinding (bottom). [ Figure 58A ] Shows the vulcanization test after 2 hours at 130°C. The sample passed the test with a color change of 4/5. [ Figure 58B ] Shows the sample left in the oven overnight after the vulcanization test. The sample still passed the test with a color change of 4/5. [ Figure 59A ] Shows the paint resistance of leather treated with 072-1 after spraying and before ironing. [ Figure 59B ] Shows the paint resistance of leather treated with 072-2 after spraying and before ironing. [ Figure 59C ] Shows the paint resistance of leather treated after spraying with 072-3 and before ironing. [ Figure 60 ] Shows the IR spectrum of the sample obtained by the LN-MCT detector. [ Figure 61 ] Shows macroscopic ATR imaging of the sample with an adhesive basecoat. [ Figure 62 ] Shows macroscopic ATR imaging of the sample with a topcoat. [ Figure 63 ] Shows a cross-section of uncoated leather. Surface unevenness is visible. [ Figure 64 ] Shows a cross-section of leather coated with a basecoat. [ Figure 65 ] Shows coated leather with the L1 system. [ Figure 66 ] Shows a further magnification of the silver marker thread in the L1 system. The bottom/top coat complex is indicated by a silver marker thread throughout the coat. [ Figure 67 ] Schematic diagram showing the resulting layers. [ Figure 68 ] Ion exchange fractionation separation scheme used to separate populations comprising low- and medium-sled/modified polypeptide compositions. Low- and medium-sled/modified polypeptide compositions contain negatively, positively, or neutrally charged silk/modified polypeptides. These populations were separated using Q anion exchange chromatography (A). [ Figure 69 ] Chromatogram of low-sled/modified polypeptide compositions loaded on a Q-Sepharose HP column (Cytiva). The flow-through contains silk/modified peptides that were not captured on the column and are depleted of negatively charged amino acids. After the column was loaded with the low-sled or medium-sled silk/modified peptide composition and the flow-through was collected, the column was washed until the UV-280 absorbance was less than 200 AU. The captured negatively charged silk/modified peptides were eluted with high salt concentration (1 M NaCl) and constituted AS11 and AS22. The chromatography was performed in a Tris-containing buffer, but the flow-through and Q-solution were ultimately dialyzed against water. [ Figure 70 ] Analytical size exclusion chromatography of the low-sled, medium-sled/modified silk compositions, and their constituent AS compositions. The average molecular weight in kDa and polydispersity measurements are shown. [ Figure 71 ] Analytical size exclusion analysis of low-sled and medium-sled/modified peptide compositions and their components (see Table 80 for more details). A , Molecular weights of various novel activated silk compositions described in this study. B , Polydispersity (PDI) of various novel activated silk compositions described in this study. AS24 reconstructs the average molecular weight and polydispersity of low-sled/modified peptide compositions and is composed of 50% AS12 and 50% AS22 (see Table 80 for details). AS6 reconstructs the average molecular weight and polydispersity of medium-sled/modified peptide compositions and is composed of 50% AS1 and 50% AS11 (see Table 80 for details). [ Figure 72 ] Isoelectric focusing of low-sled/modified peptide compositions. Lanes 2 and 7; low-sled, varying loading levels. Lanes 3, 5, 8, and 10: AS12 silk, prepared by different methods and at different loading levels. Lanes 4, 6, 9, and 11: AS22 silk, prepared by different methods and at different loading levels. [ Figure 73 ] Self-assembly reactions of low- and medium-sled silk/modified peptide combinations and their components (see Table 80 for more details). Both figures depict the kinetic parameters of gel formation during silk self-assembly. In Panel A, the calculated results of three self-assembly kinetic parameters are shown: t0.5, A max , and SARF. For more details, see the text. [ Figure 74 ] Self-assembly kinetics of low- and medium-sled silk/modified peptide combinations and their components (see Table 80 for more details). A , Self-assembly rate factor shows how quickly the self-assembly reaction is initiated and the self-assembly cores are organized to undergo self-assembly. B , Maximum gel yield shows the density of the silk gel after self-assembly is complete. C , Time required for the self-assembly reaction to produce half of the maximum amount of gel. [ Figure 75 ] Low-sled silk and medium-sled silk/modified peptide compositions and their components (see Table 80 for more details). The self-assembly factor reflects the average tendency of silk to self-assemble and form a gel. Although the above figures illustrate the embodiments disclosed in the present invention, other embodiments are also contemplated, as noted in the discussion. The present disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art, and such modifications and embodiments fall within the scope and spirit of the principles of the disclosed embodiments. [ Figure 76 ] is a graph showing the molecular weight of silk fibroin dissolved in 9.3 M LiBr at 100°C-103°C, plotted as a function of time. [ Figure 77 ] is a graph showing the molecular weight of silk fibroin dissolved in 9.3 M LiBr at 122°C-125°C, plotted as a function of time. [ Figure 78 ] is a graph showing the percentage of amino acid modifications in silk. [ Figures 79A-79C ] are graphs showing the percentage of amino acid modifications in low-sled and medium-sled silk. Figure 79A shows heavy chain modifications, Figure 79B shows light chain modifications, and Figure 79C shows fiber hexamer modifications. N is asparagine converted to aspartic acid and Q is deaminated glutamine. M corresponds to oxidized methionine. [ Figures 80A-80B ] are graphs showing the percentage of amino acid modifications in low-sled and medium-sled filaments produced and freeze-dried. Figure 80A shows heavy chain modifications, and Figure 80B shows light chain modifications. N is asparagine converted to aspartic acid and Q is deaminated glutamine. M corresponds to oxidized methionine. [ Figures 81A-81B ] are graphs showing the percentage of amino acid modifications in low-sled filaments produced in two different facilities. Figure 81A shows heavy chain modifications, and Figure 81B shows light chain modifications. N is asparagine converted to aspartic acid and Q is deaminated glutamine. M corresponds to oxidized methionine. [ Figure 82 ] is a graph showing the percentage of amino acid modifications in silk produced at sled and benchtop scales. N is asparagine converted to aspartic acid and Q is deaminated glutamine. M corresponds to oxidized methionine. [ Figure 83 ] is an explanation of the method used to calculate the percentage ratio of modified amino acids at specific positions along the sequence of each peptide. [ Figure 84 ] shows the anion exchange chromatography and size exclusion chromatography scheme for separating low-sled silk/modified peptide combinations. The low-sled/modified peptide composition is composed of diverse peptide populations covering a wide range of sizes and charges. The different populations of the low-sled/modified peptide composition were separated using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step. The Q-Sepharose eluate was loaded onto the HiLoad Superdex 200 size exclusion chromatography, which produced size-fractionated negatively charged filament compositions/modified peptides. [ Figures 85A and 85B ] show chromatograms of anion exchange chromatography and subsequent size exclusion chromatography of an eluate (Q-eluate) of the low-sled/modified peptide composition. Figure 85A : Anion exchange chromatography using a Q-Sepharose column. Anion exchange chromatography separated the low-sludge silk/modified peptide complex into an uncharged peptide population (flowthrough - light blue background) and an eluted negatively charged silk complex (eluent - light pink background). The light yellow background indicates column wash with 50 mM Tris pH 8.0 prior to elution of the charged peptide population. Figure 85B : The negatively charged eluent was loaded onto a Superdex 200 column and passed through the column with 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-sled silk/modified peptide compositions by size. The relative elution volumes of silk compositions AS77 and AS81 are indicated on the chromatograms. [ Figures 86A and 86B ] Analytical size exclusion chromatography of the low-sled silk/modified silk compositions and their component AS compositions is shown. Figure 86A shows the average molecular weight in kDa for low-sled silk (LS) and AS77-AS81. Figure 86B shows the polydispersity index (PDI) measurements. Numerical data are presented in Table 86. [ Figure 87 ] SDS-polyacrylamide gel electrophoresis of the low-sled silk/modified peptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, and their corresponding silk compositions are: Fraction 6 is AS77, Fraction 7 is AS78, Fraction 8 is AS79, Fraction 9 is AS80, and Fraction 10 is AS81. [ Figures 88A and 88B ] are graphs showing the self-assembly reaction of a low-sled silk/modified peptide combination. A medium-sled silk reaction served as a positive control. Figure 88A shows the kinetic parameters of gel formation during silk self-assembly. Self-assembly parameters for the medium-sled silk: Amax is 0.6780 (Abs), SARF is 8.676, T0.5 is 3.668 h, and FSAF is 3.08 (Abs/min). Figure 88B . A snapshot of the same self-assembly assay at a later time point, 12 days after setting up the assay. None of the fractions tested self-assembled over time. [ Figures 89A and 89B ] Characterization of low-sled silk compositions by dynamic light scattering. Low-sled silk/modified peptide compositions were diluted to a concentration of 1 mg/mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter size of each silk composition. Figure 89A . Plot of the intensity-diameter size distribution measured for silk compositions AS77, AS78, AS79, AS80, and AS81. Figure 9B . Plot of the correlation function for silk compositions AS77, AS78, AS79, AS80, and AS81. Figure 90 illustrates the size exclusion chromatography (SICA) process for separating a low-sled/modified peptide complex. The low-sled/modified peptide complex consists of diverse peptide populations spanning a wide range of sizes. Size exclusion chromatography (SICA) on a HiLoad Superdex 200 was used to separate the different populations within the complex. Figure 91 shows a chromatogram of the low-sled/modified peptide complex loaded onto a Superdex 200 gel filter column. The low-sled/modified peptide complex was loaded onto a Superdex 200 column and passed through the column with 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-sled/modified peptide complex by size. The relative solubility volumes of silk compositions AS82, AS86, and AS87 are indicated on the chromatograms. [ Figures 92A and 92B ] Analytical size exclusion chromatography of low-sled silk/modified silk compositions and their constituent AS compositions is shown. Figure 92A . Plots the average molecular weight in kDa for low-sled silk (LS) and AS82-AS89. Figure 92B . Shows polydispersity index (PDI) measurements. Numerical data are presented in Table 88. [ Figure 93 ] SDS-polyacrylamide gel electrophoresis of low-sled silk/modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, and their corresponding silk compositions are: Fraction 6 is AS82, Fraction 7 is AS83, Fraction 8 is AS84, Fraction 9 is AS85, and Fraction 10 is AS86. [ Figures 94A and 94B ] are graphs showing the self-assembly reaction of a low-sled silk/modified peptide combination. A medium-sled silk reaction served as a positive control. Figure 94A shows the kinetic parameters of gel formation during silk self-assembly. Self-assembly parameters for the medium-sled silk: Amax is 0.6978 (Abs), SARF is 8.591, T0.5 is 3.361 h, and FSAF is 3.46 (Abs/min). Figure 94B is a snapshot of the same self-assembly assay at a later time point, 18 days after setting up the assay. AS87, AS88, and AS89 exhibited gel formation at this time point, which was already observed five days after the assay (LS, low-sled silk; MS, medium-sled silk). [ Figures 95A-5C ] are graphs showing the characterization of the low-sled silk compositions by dynamic light scattering. The low-sled silk/modified peptide compositions were diluted to a concentration of 1 mg/mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 95A shows the intensity-particle size distribution measured for silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. Figure 95B shows the intensity distribution of the silk composition AS82, the low-sled silk/modified peptide composition (LS), and the medium-sled silk/modified peptide composition (MS). Figure 95C shows the correlation function of the silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, AS89, the low-sled silk/modified peptide composition (LS), and the medium-sled silk/modified peptide composition (MS). [ Figure 96 ] shows the anion exchange chromatography (Q), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC) procedures for the separation of the low-sled silk/modified peptide composition. The low-sled silk/modified peptide composition is composed of a diverse population of peptides with a wide range of sizes and charges. Different populations of low-sludge silk/modified peptide compositions were separated using Q-Sepharose anion exchange chromatography as the first step, Butyl ImpRes hydrophobic interaction resin as the second step, and HiLoad Superdex 200 size exclusion chromatography as the third purification step. The Q-Sepharose eluate was loaded onto a Butyl ImpRes (HIC) column, and the HIC eluate was loaded onto a HiLoad Superdex 200 size exclusion column. This allowed for size fractionation of negatively charged silk compositions/modified peptides with hydrophobic characteristics. The Q-Sepharose eluate contained negatively charged peptides of all sizes. Resolving these peptides on a Butyl ImpRes column results in the elution of high molecular weight, negatively charged, slightly hydrophobic silk/modified peptide compositions. Smaller negatively charged peptides are washed away as flow-through and do not bind to the Butyl ImpRes column. The Q-HIC (eluate) is loaded onto a Superdex 200 and separated by size. [ Figures 97A-97E ] Chromatograms of low-sludge silk/modified peptide compositions following anion exchange chromatography, hydrophobic interaction chromatography, and subsequent size exclusion chromatography. Figure 97A . Anion exchange chromatography using a Q-Sepharose column. A low-charge silk/modified peptide complex was separated by anion exchange chromatography into an uncharged peptide population (flowthrough - light blue background) and an eluted negatively charged silk complex (eluent - light pink background). The light yellow background indicates column washes with 50 mM Tris pH 8.0 prior to elution of the charged peptide population. Figure 97B . The negatively charged eluent (Q-eluent) was loaded onto a Butyl ImpRes column in the presence of 300 mM ammonium sulfate [(NH4)2SO4] to expose hydrophobic domains of the silk peptides, which bind to the column. The highly charged peptide population, which does not bind to the column (flowthrough), is highlighted in light blue. The column was washed until the OD280 dropped to approximately 100 units (light yellow). The bound silk peptide (Q-HIC(eluate)) was then eluted using 50 mM Tris, pH 8.0, without ammonium sulfate (light pink). Figure 97C shows further fractionation of Q-HIC(eluate) by size exclusion chromatography (SEC) using a Superdex 200 gel filter column. The Q-HIC(eluate) fraction was passed through the column using 50 mM Tris, 200 mM CaCl2, pH 8.0. When the UV-280 absorbance began to increase, the fractions were collected to separate the low-density silk/modified peptide complexes by size. The relative elution volumes of silk compositions AS90 and AS94 are indicated on the chromatogram. Figure 97D . The Q-HIC (flow-through) fraction was further fractionated by SEC using a Superdex 200 column, following the same procedure as in (VC). The relative elution volumes of silk compositions AS95 and AS100 are indicated on the chromatogram. Figure 97E . An overlay of chromatograms (VC) and (VD) is shown. The Q-HIC (flow-through) fraction has a higher molecular weight range than the Q-HIC (flow-through) fraction, which elutes later in SEC and has a lower molecular weight range. Figures 98A-98B show analytical size exclusion chromatography of low-sled silk/modified silk compositions and their constituent AS compositions. Figure 98A shows the average molecular weight in kDa for low-sled silk (LS) and AS90-AS100. Figure 98B shows polydispersity index (PDI) measurements. Numerical data are presented in Table 90. Figures 99A-99B show SDS-polyacrylamide gel electrophoresis of low-sled silk/modified polypeptide compositions. Figure 99A shows Q-HIC (eluate) SEC fractions. Figure 99B shows Q-HIC (flowthrough) SEC fractions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, and their corresponding silk compositions: In Figure 99A , fraction 6 is AS90, fraction 7 is AS91, fraction 8 is AS92, fraction 9 is AS93, and fraction 10 is AS94. In Figure 99B , fraction 8 is AS95, fraction 9 is AS96, fraction 10 is AS97, fraction 11 is AS98, fraction 12 is AS99, and fraction 13 is AS100. [ Figure 100 ] shows the self-assembly reaction of a low-sled silk/modified peptide combination. A medium-sled silk reaction served as a positive control. Kinetic parameters of gel formation during silk self-assembly. Q-HIC (eluate) is the fraction eluted from the Butyl ImpRes column before SEC purification; LS, low-sled silk; MS, medium-sled silk. Self-assembly parameters for the medium-sled silk: Amax of 0.6974 (Abs), SARF of 8.661, T0.5 of 3.834 h, and FSAF of 3.03 (Abs/min). [ Figures 101A-101F ] Characterization of the low-sled silk composition by dynamic light scattering. The low-sled silk and medium-sled silk/modified peptide compositions were diluted to a concentration of 1 mg/mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 101A shows the measured intensity-diameter particle size distribution for silk composition AS90, the Q-HIC (eluate) fraction (before fractionation by SEC), the low sled wire (LS), and the medium sled wire (MS). Figure 101B shows the correlation function for the silk composition presented in (ZA). Figure 101C shows the measured intensity-diameter particle size distribution for silk compositions AS90-AS94 resulting from the Q-HIC (eluate)-SEC fractionation process. Figure 101D shows the correlation function for the silk composition presented in (ZC). Figure 101E shows the measured intensity-diameter particle size distribution for silk compositions AS95-AS100 derived from a Q-HIC (flowthrough)-SEC fractionation process. Figure 101F shows the correlation function for the silk compositions presented in (ZE). [ Figure 102 ] illustrates the size exclusion chromatography process for separating mid-sled silk/modified peptide compositions. Mid-sled silk/modified peptide compositions consist of diverse peptide populations spanning a wide range of sizes. Size exclusion chromatography using a HiLoad Superdex 200 allows for the separation of distinct populations within mid-sled silk/modified peptide compositions. [ Figure 103 ] shows a chromatogram of mid-sled silk/modified peptide compositions loaded on a Superdex 200 gel filter column. The mid-sled silk/modified peptide combination was loaded onto a Superdex 200 column and passed through with 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the mid-sled silk/modified peptide combination by size. The relative elution volumes of silk compositions AS107 and AS111 are indicated on the chromatogram. [ Figures 104A-104B ] Analytical size exclusion chromatography of the mid-sled silk/modified silk combination and its constituent AS compositions is shown. Figure 104A shows the average molecular weight in kDa for mid-sled silk (MS) and AS106-AS111. Figure 104B shows the polydispersity (PDI) measurements. Numerical data are presented in Table 92. [ Figure 105 ] shows SDS-polyacrylamide gel electrophoresis of a mid-sled filament/modified peptide composition. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, and the corresponding filament compositions are: Fraction 6 is AS107, Fraction 7 is AS108, Fraction 8 is AS109, Fraction 9 is AS110, and Fraction 10 is AS111. [ Figure 106 ] shows the self-assembly reaction of a mid-sled filament/modified peptide composition. Kinetic parameters for gel formation during filament self-assembly. The red dashed line shows how the self-assembly parameters Amax, SARF, and T0.5 of the mid-sled filament (MS) were calculated from the unfractionated separation. The numerical calculation parameters for silk compositions AS106-AS111 can be found in Table 93. Low-sled silk (LS) served as a negative control. LS, low-sled silk; MS, medium-sled silk. [ Figures 107A-107B ] Characterization of medium-sled silk compositions by dynamic light scattering is shown. Medium-sled silk/modified peptide compositions were diluted to a concentration of 1 mg/mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 107A . Intensity-particle size distribution measured for silk compositions AS106, AS107, AS108, AS109, AS110, AS111, and medium-sled (MS). Figure 107B . Correlation function for the silk compositions presented in (A). Figure 108 illustrates the anion exchange chromatography and size exclusion chromatography workflow for the separation of mid-sled filaments/modified peptide compositions. Mid-sled filaments/modified peptide compositions consist of diverse peptide populations spanning a wide range of sizes and charges. Q-Sepharose anion exchange chromatography was used as the first step, and HiLoad Superdex 200 size exclusion chromatography was used as the second purification step to separate the different populations of mid-sled filaments/modified peptide compositions. The Q-Sepharose eluate was loaded onto the HiLoad Superdex 200 size exclusion chromatography, which yielded size-fractionated negatively charged filament compositions/modified peptides. Figures 109A-109B show anion exchange chromatography (ACE) and subsequent size exclusion chromatography (SEC) of the eluate (Q-Sepharose) of a mid-sled filament/modified peptide complex. Figure 109A . Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). ACE separation of the mid-sled filament/modified peptide complex into an uncharged peptide population (flowthrough - light blue background) and an eluted negatively charged filament complex (eluate - light pink background) was performed. The light yellow background indicates column wash with 50 mM Tris pH 8.0 prior to elution of the charged peptide population. Figure 109B . The negatively charged eluate (Q-eluate) was loaded onto a Superdex 200 column and passed through with 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the mid-sled silk/modified peptide complex by size. The relative eluate volumes of silk complexes AS101 and AS105 are indicated on the chromatogram. [ Figures 110A-110B ] Analytical size exclusion chromatography of the mid-sled silk/modified silk complex and its constituent AS complexes is shown. Figure 110A . Shows the average molecular weight in kDa for mid-sled silk (MS) and AS101-AS105. Figure 110B shows polydispersity (PDI) measurements. The numerical data are presented in Table 95. Figure 111 shows SDS polyacrylamide gel electrophoresis of a mid-sled silk/modified peptide composition. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, and the corresponding silk compositions are: Fraction 6 is AS101, Fraction 7 is AS102, Fraction 8 is AS103, Fraction 9 is AS104, and Fraction 10 is AS105. Figure 112 shows the self-assembly reaction of a mid-sled silk/modified peptide composition. A low-sled silk reaction was used as a negative control. The kinetic parameters of gel formation during silk self-assembly are shown. Dashed red lines are shown to illustrate the calculation of the Amax, SARF (self-assembly rate factor), and T0.5 parameters in Table 96. [ Figures 113A-113C ] Graphs depicting the characterization of the sled silk compositions by dynamic light scattering. The sled silk/modified peptide compositions were diluted to a concentration of 1 mg/mL, filtered, and analyzed using a Zetasizer Pro (Malvern) to estimate the diameter particle size of each silk composition. Figure 113A . Diameter particle size distribution by intensity measured by the intensometer for silk compositions AS101, AS102, AS103, AS104, and AS105. Figure 113B . Particle size distribution by intensometer intensity diameter measured for silk compositions AS101, AS105, and a medium sled wire (MS), highlighting the size difference between AS101 and AS105. Figure 113C. Correlation plot function for silk compositions AS101, AS102, AS103, AS104, AS105, and a medium sled wire (MS). [ Figure 114 ] Plot of the values of the three molar mass moments (Mn, Mw, and Mz) as they relate to molar mass and the number of molecules per molar mass. This example applies to polydisperse samples; for monodisperse samples, Mn = Mw = Mz. [ Figures 115A-115B ] Analytical SEC-MALS of low-, medium-, and high-molecular-weight silks. Figure 115A . Weight average molecular weight in kDa for low, medium, and high molecular weight silks. Figure 115B . Polydispersity index (PDI) measurements for low, medium, and high molecular weight silks are shown. [ Figures 116A-116B ] Analytical SEC-MALS of low, medium, and high molecular weight silks organized by silk type. Individual data points are shown, and the mean is represented by the height of the box. Bars cover one standard deviation. Figure 116A . Weight average molecular weight range for low, medium, and high molecular weight silks. Figure 116B . PDI range for low, medium, and high molecular weight silks. [ Figures 117A-117B ] Analytical SEC-MALS of a low-sled silk/modified silk composition and a constitutive AS composition separated by Q-SEC (Q-solvent). Figure 117A shows the average molecular weight in kDa for low-sled silk (LS) and AS77-AS81. Figure 117B shows the polydispersity (PDI) measurements. The numerical data are presented in Table 103. Figures 118A-118B show analytical SEC-MALS of a low-sled silk/modified silk composition and a constitutive AS composition separated by SEC. Figure 118A shows the average molecular weight in kDa for low-sled silk (LS) and AS82-AS89. Figure 118B shows the polydispersity (PDI) measurements. The numerical data are presented in Table 104. Figures 119A-119B show analytical SEC-MALS analysis of a low-sled silk/modified silk composition and a constitutive AS composition separated by Q-HIC-SEC (Q-HIC-eluent). Figure 119A shows the average molecular weight in kDa for the low-sled silk (LS) and AS90-AS94. Figure 119B shows the polydispersity index (PDI) measurements. Numerical data are presented in Table 105. Figures 120A-120B show analytical SEC-MALS analysis of a low-sled silk/modified silk composition and a constitutive AS composition separated by Q-HIC-SEC (Q-HIC-flowthrough). Figure 120A shows the average molecular weight in kDa for the low-sled silk (LS) and AS95-AS100. Figure 120B shows polydispersity (PDI) measurements. The numerical data are presented in Table 105. [ Figures 121A-121B ] show analytical SEC-MALS of a mid-sled/modified silk composition and a constitutive AS composition separated by Q-SEC (Q-flowthrough). Figure 121A shows the average molecular weight in kDa for mid-sled silk (MS) and AS101-AS105. Figure 121B shows polydispersity (PDI) measurements. The numerical data are presented in Table 106. [ Figures 122A-122B ] show analytical SEC-MALS of a mid-sled/modified silk composition and a constitutive AS composition separated by SEC. Figure 122A shows the average molecular weight in kDa for medium sled wire (MS) and AS106-AS111. Figure 122B shows the polydispersity index (PDI) measurements. The numerical data are presented in Table 107. [ Figures 123A-123B ] are SEM images of unpainted leather. [ Figures 124A-124B ] are SEM images of leather with a silver-labeled primer. Leather sprayed with a 4 gr/sf silver-labeled primer. [ Figures 125A-125B ] are SEM images of leather sprayed with a 4 gr/sf silver-labeled primer. Leather sprayed with a 4 gr/sf silver-labeled primer at higher magnification. Figure 126 shows an SEM image of a silver-marked, fully L1-finished surface (base coat + top coat). The leather was sprayed with a 4 gr/sf silver-marked base coat and a 6 gr/sf top coat. This is a side view, rotated 45°. Figures 127A-127B show an SEM image of a silver-marked, fully L1-finished surface (base coat + top coat). The leather was sprayed with a 4 gr/sf silver-marked base coat and a 6 gr/sf top coat. This is a cross-sectional view. [ Figure 128 ] is a photograph showing an example of a failed milled leather (left) and a passed milled leather (right). [ Figure 129 ] illustrates the process of creating a pattern on leather using paper peeling. [ Figure 130 ] illustrates the process used to create a pattern on leather with large defects or blemishes. [ Figures 131A-131B ] are illustrations of the final composition of leather products made using paper peeling transfer leather with an Activated Silk™ top coat (Figure 131A) and a traditional top coat (Figure 131B). The Activated Silk™ top coat has a lower solids concentration than the traditional top coat (1.5-3% vs. 15-35%). When applied to an Activated Silk™ L1 basecoat, it creates a composite. [ Figure 132 ] is an image of a low-grade leather before the Activated Silk™ L1 paper peel treatment. [ Figure 133 ] is an image of a low-grade leather after the Activated Silk™ L1 paper peel treatment. [ Figure 134 ] is a close-up image of a low-grade leather after the Activated Silk™ L1 paper peel treatment. [ Figure 135 ] is a cross-sectional view of an uncoated crust leather. [ Figure 136 ] is a cross-sectional view of a crust leather with a laminate (pre-skin and skin). [ Figure 137 ] is a cross-sectional view of a crust leather with a laminate and basecoat. Figure 138 shows a cross-sectional view of a crust leather with a laminated film (front and top skin) and a base coat. Figure 139 shows a cross-sectional view of a crust leather with a laminated film (front and top skin) and a base coat. Figure 140 shows a cross-sectional view of a crust leather with a laminated film (front and top skin) and a base coat. Figure 141 shows a cross-sectional view of the finished leather with a laminated film, a base coat, and a top coat after rotary pressing.

Although the above drawings illustrate embodiments of the present invention, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art that fall within the scope and spirit of the principles of the present invention.

TW202528611A_113133963_SEQL.xml

Claims (98)

An article comprising a substrate and a coating, wherein the coating comprises a base layer comprising a first polymeric macromolecular species or polymer and a top layer comprising a second polymeric macromolecular species or polymer, wherein the base layer comprises mechanically engineered topographical features on a surface opposite the substrate, wherein the engineered topographical features have width and/or depth dimensions on the scale of about 0 µm to about 250 µm, wherein a portion of the base layer and a portion of the top layer form a composite layer that substantially represents and/or retains the topographical features of the base layer. The article of claim 1, further comprising one or more additional layers disposed between the substrate and the base layer, the additional layers being selected from a pre-polishing layer, a polishing layer, and an adhesive layer. The article of claim 2, wherein the base layer topography is substantially different from any unengineered topography of the substrate, the pre-polishing layer, the polishing layer, and/or the adhesive layer. The article of claim 2 or 3, wherein the composite layer topography is substantially different from any unengineered topography of the substrate, the pre-polishing layer, the polishing layer, and/or the adhesive layer. The article of any one of claims 1 to 4, wherein the composite layer comprises a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer. The product of any one of claims 1 to 5, wherein the mechanically engineered morphological features include one or more of mechanically engineered embossed features, mechanically engineered relief features, mechanically engineered recessed features, mechanically engineered stamped features and/or mechanically engineered repetitive features. The article of any one of claims 1 to 5, wherein the mechanically engineered topographical features comprise one or more of mechanically engineered embossed features and/or mechanically engineered imparted patterns. The article of any one of claims 1 to 5, wherein the mechanically engineered topographical features comprise one or more mechanically engineered embossed particle patterns. The article of any one of claims 1 to 5, wherein the mechanically engineered topographical features comprise one or more mechanically engineered embossed fine particle features having a particle depth between 0-100 μm. The article of any one of claims 1 to 5, wherein the mechanically engineered topographical features comprise one or more mechanically engineered embossed coarse grain features having a grain depth between 100-150 μm. The article of any one of claims 1 to 10, wherein the composite layer comprises a portion of the base layer and a portion of the top layer, wherein a portion of the base layer and a portion of the top layer are physically and/or chemically entangled, and/or physically and/or chemically cross-linked, and/or chemically and/or physically integrated. The article of any one of claims 4 to 11, wherein the adhesive layer comprises one or more of the following and/or is produced from one or more of the following: acrylic dispersion, polyurethane dispersion, aqueous urethane-acrylic hybrid dispersion (HPDS), wax, oil-in-water emulsion and/or polysiloxane. The article of any one of claims 1 to 12, wherein the first polymeric macromolecular species or polymer comprises a protein component. The product of claim 13, wherein the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein and/or whey. The article of any one of claims 1 to 13, wherein the base layer comprises one or more of the following and/or is produced from one or more of the following: a polyurethane dispersion, a wax, an oil-in-water emulsion and/or a protein binder. The article of any one of claims 1 to 13, wherein the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component and/or a poly(lactic-co-glycolic acid) (PLGA) component. The article of any one of claims 1 to 16, wherein the first polymeric macromolecular species or polymer comprises a biodegradable polymer. The article of any one of claims 1 to 17, wherein the base layer has a thickness between about 10 μm and about 35 μm. The article of any one of claims 1 to 18, wherein the second polymeric macromolecular species or polymer comprises one or more of a cellulose derivative, an aliphatic or aromatic polyurethane, a silanol/amino-polysiloxane emulsion, a cross-linked polyurethane, treated silica, and/or a protein component. The product of claim 19, wherein the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein and/or whey. The article of any one of claims 1 to 20, wherein the second polymeric macromolecular species or polymer comprises a biodegradable polymer. The article of any one of claims 1 to 21, wherein the second polymeric macromolecular species or polymer comprises cellulose and/or a cellulose derivative component. The product of claim 22, wherein the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate and microcrystalline cellulose. The product of claim 23, wherein the cellulose derivative is ethyl cellulose. The product of claim 23 or 24, wherein the ethoxy content of the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%. The product of claim 24 or 25, wherein the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3. The article of any one of claims 24 to 26, wherein the second structure of the cellulose derivative comprises less than 100%, between about 5% and less than about 100%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 99%, or between about 90% and about 100% crystallinity. The article of any of claims 24 to 26, wherein the second structure of the cellulose derivative comprises less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, Less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 58%, less than about 57% , less than about 56%, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33 %, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10% crystallinity. The article of any one of claims 1 to 28, wherein the top layer has a thickness between about 5 μm and about 10 μm. The article of any one of claims 1 to 29, wherein the thickness ratio of the base layer to the top layer is in a range of about 10:1 to about 1:1. The article of any one of claims 1 to 30, wherein the top layer does not include an adhesive material or a crosslinking agent. The article of any one of claims 1 to 31, wherein the first polymeric macromolecular species or polymer is anisotropically distributed across a cross section of the composite layer. The article of any one of claims 1 to 31, wherein the second polymeric macromolecular species or polymer is anisotropically distributed across a cross section of the composite layer. The article of any one of claims 1 to 33, wherein the substrate comprises an irregular surface. The article of any one of claims 1 to 33, wherein the substrate comprises topographical features that have not been mechanically engineered. The article of any one of claims 1 to 35, wherein the coating has a thickness between about 10 μm and about 1000 μm. The article of any one of claims 1 to 36, wherein the amount of coating on the substrate is between about 0.01 g/ ^ft2 and about 25 g/ ^ft2 . The article of any one of claims 1 to 37, wherein the substrate comprises a substantially flexible material. The article of any one of claims 1 to 38, wherein the substrate comprises a leather material or a textile material. The article of any one of claims 1 to 39, wherein the substrate comprises one or more of collagen, fibrous protein, keratin, cellulose and/or lignin. The article of any one of claims 1 to 40, wherein the coating comprises one or more matting agents. The article of any one of claims 1 to 40, wherein the coating comprises one or more plasticizers. The article of any one of claims 1 to 42, wherein the coating comprises a plurality of modified fibroin fragments, each of which comprises one or more amino acid residue modifications selected from the group consisting of an asparagine to aspartic acid modification, a glutamine to glutamine modification, and a methionine to methionine oxide modification. The article of claim 43, wherein the plurality of modified fibroin segments comprise a modification. The article of claim 43, wherein the plurality of modified fibroin segments comprises two modifications. The article of claim 43, wherein the plurality of modified fibroin segments comprises three modifications. The product of any of claims 43 to 46, wherein the asparagine to aspartic acid modification is at one or more positions selected from the group consisting of: N23, N28, N108, N118, N136, N186, N200, N204, N240, N248, N68, N70, N77, N5262, N93, N132, N149, N172, N174, N202, N105, N4191. The product of any one of claims 43 to 46, wherein the glutamine to glutamine modification is at one or more positions selected from the group consisting of Q24, Q149, Q202, Q58, Q139, Q275, Q5216, Q255, and Q125. The product of any one of claims 43 to 46, wherein the methionine to methionine oxide modification is at position M64. The article of any one of claims 43 to 49, wherein each modification independently comprises between about 1% and about 99% of the composition. The article of any one of claims 43 to 50, wherein % modification is defined as (the number of modified silk fibroin segments having a modification at a specific position divided by the total number of modified silk fibroin segments including that specific position, whether modified or unmodified)×100. A method for coating a substrate with a composite coating, the method comprising: applying a base coating composition to the substrate by a release paper method, wherein the release paper forms a plurality of mechanically engineered topographical features on the base layer opposite the surface applied to the substrate, wherein the engineered topographical features have width and/or depth dimensions on the scale of about 0 μm to about 250 μm; and applying a top coating composition. The method of claim 52, further comprising applying one or more additional layers of coating compositions to the substrate before applying the base layer coating composition, the additional layers being selected from a pre-polishing layer, a polishing layer, and an adhesive layer. The method of claim 52 or 53, wherein the adhesive coating composition comprises one or more of an acrylic dispersion, a polyurethane dispersion, an aqueous urethane-acrylic hybrid dispersion (HPDS), a wax, an oil-in-water emulsion, and/or a polysiloxane. The method of any one of claims 52 to 54, wherein the base coating composition comprises a protein component. The method of claim 55, wherein the protein component comprises one or more of fibrous protein or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein, and/or whey. The method of any one of claims 52 to 56, wherein the base coating composition comprises one or more of a polyurethane dispersion, a wax, an oil-in-water emulsion, and/or a protein binder. The method of any one of claims 52 to 56, wherein the base coating composition comprises a polylactic acid (PLA) component and/or a poly(lactic-co-glycolic acid) (PLGA) component. The method of any one of claims 52 to 56, wherein the base coating composition comprises a biodegradable polymer. The method of any one of claims 52 to 59, wherein the top coating composition comprises one or more of a cellulose derivative, an aliphatic or aromatic polyurethane, a silanol/amino-polysiloxane emulsion, a cross-linked PU, treated silica, and/or a protein component. The method of claim 60, wherein the top coating composition comprises one or more of silk fibroin or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein, and/or whey. The method of any one of claims 52 to 61, wherein the top coating composition comprises a biodegradable polymer. The method of any one of claims 52 to 61, wherein the top coating composition comprises cellulose and/or a cellulose derivative component. The method of claim 63, wherein the cellulose derivative is selected from methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. The method of claim 63, wherein the cellulose derivative is ethyl cellulose. The method of claim 64 or 65, wherein the ethoxy content of the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%. The method of claim 65 or 66, wherein the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3. The method of any one of claims 65 to 67, wherein the second structure of the cellulose derivative comprises less than 100%, between about 5% and less than about 100%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 99%, or between about 90% and about 100% crystallinity. The method of any of claims 65 to 67, wherein the second structure of the cellulose derivative comprises less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, Less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 58%, less than about 57% , less than about 56%, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33 %, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10% crystallinity. The method of any of claims 52 to 69, wherein the substrate comprises a substantially flexible material. The method of any one of claims 52 to 69, wherein the substrate comprises a leather material or a textile material. The method of any one of claims 52 to 69, wherein the substrate comprises one or more of collagen, fibrous protein, keratin, cellulose, and/or lignin. The method of any one of claims 52 to 72, wherein the coating composition comprises a matting agent. The method of any one of claims 52 to 72, wherein the coating composition comprises a plasticizer. The method of any one of claims 52 to 74, wherein the coating composition comprises a plurality of modified fibroin fragments, each of which comprises one or more amino acid residue modifications selected from the group consisting of an asparagine to aspartic acid modification, a glutamine to glutamine modification, and a methionine to methionine oxide modification. The method of claim 75, wherein the plurality of modified fibroin fragments comprise one modification. The method of claim 75, wherein the plurality of modified fibroin fragments comprises two modifications. The method of claim 75, wherein the plurality of modified fibroin fragments comprises three modifications. The method of any of claims 75 to 78, wherein the asparagine to aspartic acid modification is at one or more positions selected from the group consisting of: N23, N28, N108, N118, N136, N186, N200, N204, N240, N248, N68, N70, N77, N5262, N93, N132, N149, N172, N174, N202, N105, N4191. The method of any one of claims 75 to 78, wherein the glutamine to glutamine modification is at one or more positions selected from the group consisting of Q24, Q149, Q202, Q58, Q139, Q275, Q5216, Q255, and Q125. The method of any one of claims 75 to 78, wherein the methionine to methionine oxide modification is at position M64. The method of any one of claims 75 to 81, wherein each modification independently comprises between about 1% and about 99% of the composition. The method of any one of claims 75 to 82, wherein % modification is defined as (the number of modified silk fibroin segments having a modification at a specific position divided by the total number of modified silk fibroin segments including that specific position, modified or unmodified)×100. The method of any one of claims 52 to 83, wherein the coating composition further comprises a solvent component. The method of claim 84, wherein the solvent component comprises alcohol and/or alcohol derivatives. The method of claim 84 or 85, wherein the solvent component comprises one or more of an alcohol, an ether, a ketone, an aldehyde and/or a ketal. The method of any one of claims 84 to 86, wherein the solvent component is about 75% w/w to about 99% w/w of the coating composition, about 80% w/w to about 98% w/w of the coating composition, about 85% w/w to about 97.5% w/w of the coating composition, or about 85% w/w to about 95% w/w of the coating composition. The method of any one of claims 84 to 87, wherein the solvent component comprises one or more of methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, pentanol, hexanol, acetone, butanone, methoxypropanol, diisopropyl glycerol, 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, 2,2-dimethyl-1,3-dioxolane-4-methanol, or any combination thereof. The method of any one of claims 52 to 88, wherein the coating composition comprises one or more of a polyethylene glycol (PEG) component, a polypropylene glycol (PPG) component, and/or a polyether component. The method of any one of claims 52 to 88, wherein the coating composition comprises a fatty acid or a fatty acid-derived amide and/or one or more of a monoglyceride, a diglyceride, and/or a triglyceride. The method of any one of claims 52 to 88, wherein the coating composition comprises one or more of a triethylene glycol monomethyl ether component, a diethylene glycol butyl ether component, a diethylene glycol ethyl ether component, a tetradecanedioic acid dimethyl ester component, an erucamide component, and/or a stearic acid glyceryl ester component. The method of any one of claims 52 to 91, wherein the coating composition comprises water. The method of any one of claims 52 to 92, wherein the coating composition comprises a matting agent. The method of any one of claims 52 to 92, wherein the coating composition comprises a plasticizer. The method of any one of claims 52 to 94, further comprising one or more pressing steps, and/or one or more drying or partial drying steps. The method of any one of claims 52 to 95, wherein the first coating composition is partially polymerized, partially dried, and/or partially cured prior to applying the second coating composition. The method of any one of claims 52 to 96, wherein the coating composition is applied at a rate of about 0.5 mL/ft ² to about 5 mL/ft ² . An article comprising a substrate and a coating, the article being produced by the method of any one of claims 52 to 97.